CN101061228B - Isopentenyl transferase sequences and methods of use - Google Patents

Abstract

Methods and compositions for modulating plant development are provided. Polynucleotide sequences and amino acid sequences encoding isopentenyl transferase (IPT) polypeptides are provided. The sequences can be used in a variety of methods including modulating root development, modulating floral development, modulating leaf and/or shoot development, modulating senescence, modulating seed size and/or weight, and modulating tolerance of plants to abiotic stress. Polynucleotides comprising an IPT promoter are also provided. The promoter can be used to regulate expression of a sequence of interest. Transformed plants, plant cell, tissues, and seed are also provided.

Description

Prenyltransferase sequences and methods of use thereof

technical field

The present invention relates to the field of genetic manipulation of plants, particularly the regulation of gene activity to affect plant development and growth.

Background of the invention

Cytokinins are a class of N6 ^- substituted purine-derived plant hormones that regulate cell division and affect a variety of developmental events, such as shoot development, sink strength, root branching, control of shoot apical dominance, leaf development , Chloroplast development and leaf senescence, etc. (Mok et al. (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, FLA, pp.155-166; Horgan (1984) Advanced Plant Physiology ed. MB., Pitman, London, UK, pp 53-75; and Letham (1994) Annual Review of Plant Physiol 34: 163-197). In maize, cytokinin (CK) plays an important role in determining seed size, reducing top grain abortion and increasing seed fixation under adverse environmental conditions (Cheikh et al. (1994) Plant Physiol. 106: 45-51; Dietrich et al. (1995) Plant Physiol Biochem 33:327-36). The pool of active cytokinins is regulated by the rate of synthesis and degradation.

Until recently, the root was thought to be the major site of cytokinin biosynthesis, but there is also evidence that other tissues, such as shoot meristems and developing seeds, also have high cytokinin biosynthetic activity. This suggests that cytokinins are synthesized at defined sites where cell proliferation is active. Several AtIPT genes present in Arabidopsis and their different expression patterns may be involved in achieving this purpose.

The catalytic enzyme isopentenyltransferase (IPT) directs the synthesis of cytokinins and plays an important role in regulating the levels of cytokinins in plant tissues. Multiple pathways for cytokinin biosynthesis have been proposed. Since some tRNA molecules contain isopentenyl adenosine (iPA) residues adjacent to the anticodon site, degradation of transfer RNA may be the source of cytokinins (Swaminathan et al. (1977) Biochemistry 16:1355 -1360). The modification is catalyzed by tRNA pentenyltransferase (tRNA IPT; EC 2.5.1.8), which has been demonstrated in bacteria such as Escherichia coli, Saccharomyces cerevisiae, Lactobacillus acidophilus, Homo sapiens (Bartz et al. (1972) Biochemie 54: 31-39; Kline et al. (1969) Biochemistry 8: 4361-4371; Holtz et al. (1975) Hoppe- Seyler's Z. Physiol. Chem 356: 1459-1464; Golovko et al. (2000) Gene 258: 85-93; Holtz et al. (1979) Hoppe-Seyler's Z. Physiol. Chem 359: 89-101). But it is not considered that this pathway is the main pathway for cytokinin synthesis (Chen et al. (1997) Physiol. Plant 101: 665-673 McGraw et al. (1995) Plant Hormones, Physiology, Biochemistry and Molecular Biology. Ed. Davies, 98 -117, Kluwer Academic Publishers, Dordrecht).

Another possible pathway for the formation of cytokinins is de novo via adenylate isopentenyltransferase (IPT; EC 2.5.1.27) with dimethylallyl diphosphate (DMAPP), AMP, ATP, and ADP as substrates Biosynthesis of iPMPs. Our current knowledge of cytokinin biosynthesis in plants has been largely inferred from studies of possible homologous systems in Agrobacterium tumefaciens. Agrobacterium tumefaciens cells can infect specific plant species by inducing neoplasia in host plant tissues (VanMontagu et al. (1982) Curr Top Microbiol Immunol 96:237-254; Hansen et al. (1999). Curr Top Microbiol Immunol 240:21-57). To this end, A. tumefaciens cells synthesize and secrete cytokinins that mediate the transformation of normal host plant tissue into tumors or calli. This process can be facilitated by the Agrobacterium tumefaciens tumor-inducing plasmid containing genes encoding enzymes and regulators necessary for cytokinin biosynthesis. Biochemical and genetic studies have shown that gene 4 of the tumor-inducing plasmid encodes isopentenyltransferase (IPT), which converts AMP and DMAPP into the active form of cytokinin, isopentenyladenosine-5'-monophosphate (iPMP) (Akiyoshi et al. (1984) Proc. Natl. Acad. Sci USA 81:5994-5998). Overexpression of the Agrobacterium ipt gene in various transgenic plants has been shown to cause increased cytokinin levels in the host plant and elicit a typical cytokinin response (Hansen et al. (1999) Curr Top Microbiol Immunol 240:21- 57). Plant cells are therefore presumed to use a cytokinin biosynthetic machinery similar to that of Agrobacterium tumefaciens. Arabidopsis IPT homologues were recently discovered in Arabidopsis and petunia (Takei et al. (2001) J. Biol. Chem. 276: 26405-26410 and Kakimoto (2001) Plant Cell Physiol. 42: 677- 685). Overexpression of Arabidopsis IPT homologues in plants increased cytokinin levels, eliciting typical cytokinin responses in plants under tissue culture conditions (Kakimoto (2001) Plant Cell Physiol.42:677-685 ).

The Arabidopsis ipt gene is a member of a small multigene family consisting of nine distinct genes, two of which encode tRNA prenyltransferases and seven of which encode gene products with cytokinin biosynthetic functions . Biochemical analysis of the recombinant AtIPT4 protein revealed that, in contrast to the bacterial enzyme, the Arabidopsis enzyme uses ATP instead of AMP as a substrate. Another plant IPT gene (Sho) was identified in Petunia hybrida using an activation tagging strategy (Zubko et al. (2002) The Plant Journal 29:797-808).

Since cytokinins affect multiple plant developmental processes including root formation, shoot and leaf development, and seed fixation, the ability to modulate cytokinin levels in higher plant cells, thereby strongly affecting plant growth and productivity, provides significant commercial value (Mok et al. (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, FLA, pp. 155-166).

brief description of the invention

The compositions and methods of the invention comprise and use isopentenyltransferase (IPT) polypeptides and polynucleotides involved in the regulation of plant development, morphology and physiological processes.

Compositions further comprise expression cassettes, plants, plant cells and seeds having the IPT sequences of the invention. Plants, plant cells and seeds of the invention may have phenotypic changes, such as modulated (increased or decreased) cytokinin levels, modulated floral Regulated root development; Changed shoot to root ratio; Increased seed size or increased seed weight; Increased plant yield or plant vigor; Maintain or improve stress tolerance (e.g., increase or maintain plant size , reduce top grain abortion, increase or maintain seed fixation); reduce shoot growth; delay senescence or enhance plant growth-related changes.

The compositions of the present invention also include IPT promoters, DNA constructs comprising the IPT promoter operably linked to a nucleic acid sequence of interest, expression vectors comprising these DNA constructs, plants, plant cells and seeds.

A method for reducing or eliminating the activity of an IPT polypeptide in a plant is provided, comprising introducing the selected polynucleotide into the plant. In particular methods, provided polynucleotides reduce cytokinin levels in a plant and/or modulate plant root development.

Also provided are methods of increasing the level of an IPT polypeptide in a plant comprising introducing a selected polyn into the plant. In particular methods, expression of the IPT polynucleotide increases cytokinin levels in plants; maintains or improves stress tolerance in plants; maintains or increases plant size; reduces seed abortion; increases or maintains seed fixation; growth; increase seed volume or grain weight; increase plant yield or vigor; regulate floral development; delay senescence or increase leaf growth.

Also provided are methods of modulating expression of a nucleic acid sequence of interest. The method comprises introducing into a plant a DNA construct comprising a heterologous nucleotide sequence of interest operably linked to the IPT promoter of the present invention.

Brief description of the drawings

Figure 1 is a sequence alignment of cytokinin biosynthetic enzymes from maize, petunia and Arabidopsis. The amino acid sequences in the alignment include ZmIPT1 (SEQ ID NO: 23), ZmIPT2 (SEQ ID NO: 2), ZmIPT4 (SEQ ID NO: 6), ZmIPT5 (SEQ ID NO: 9), ZmIPT6 (SEQ ID NO: 12) , ZmIPT7 (SEQ ID NO: 15), ZmIPT8 (SEQ ID NO: 18), AtIPT1 (SEQ ID NO: 29), AtIPT3 (SEQ ID NO: 34), AtIPT4 (SEQ ID NO: 30), AtIPT5 (SEQ ID NO : 35), AtIPT6 (SEQ ID NO: 36), AtIPT7 (SEQ ID NO: 37), AtIPT8 (SEQ ID NO: 38) and Sho (SEQ ID NO: 31). Asterisks indicate amino acids conserved in multiple IPT proteins, underlined amino acids indicate putative ATP/GTP binding sites.

Fig. 2 is a schematic structural representation of the ZmlPT1 gene (SEQ ID NO: 21) from Mo17. Coding regions are indicated by thick arrows, and the CAAT and putative TATA boxes are marked.

Fig. 3 is the amino acid sequence alignment of ZmlPT1 (SEQ ID NO: 23, expressed as ZmlPT-Mo17) and ZmlPT1 variant (SEQ ID NO: 27, expressed as ZmlPT-B73). The sequences share 98% amino acid sequence identity. A sequence identical to the ZmlPT1 polypeptide was found at SEQ ID NO:39.

Fig. 4 is the ppm value of ZmlPT1 in the Lynx embryo library at different days after pollination (DAP).

Figure 5A shows the detection of ZmlPT1 in different maize organs by RT-PCR.

Figure 5B shows the detection of ZmlPT1 in developing grains by RT-PCR.

Figure 6 shows a Southern blot of the B73 or Mo17 genome digested by 3 different restriction enzymes. 40 μg of genomic DNA was digested, electrophoresed in a 0.8% agarose gel, and transferred to a nylon membrane. The coding sequence of ZmlPT2-B73 gene was used as the probe.

Figure 7 shows the Northern blot and relative expression of ZmlPT2 gene in different plant organs and whole grains at different days after pollination (DAP). Transcript levels were measured in leaves (L), stems (S), roots (R) and whole grains at 0, 5, 10, 15, 20 and 25 days after pollination and compared to the abundance of cyclophilin transcripts. to quantify.

Figure 8 shows the Northern blotting in different days after pollination and the relative expression of ZmIPT2 gene in grains. Transcript levels were determined at 0 to 5-DAP for whole grains and 6 to 34-DAP for peduncled grains and quantified relative to the abundance of cyclophilin transcripts. The levels of zeatin nucleosides (the most abundant CK in corn kernels) in the same samples were previously determined and are represented by solid lines (Brugière et al. (2003) Plant Phsyiol 132: 1228-1240).

Figure 9 is the ppm value of ZmlPT2 in the Lynx embryo library.

Figure 10 is an alignment of the amino acid sequences corresponding to the Arabidopsis IPT protein (AtIPT), the petunia IPT protein (Sho) and the rice putative IPT protein (OsIPT). The sequences in the alignment are as follows: OsIPT6 (SEQ ID NO: 57); OsIPT8 (SEQ ID NO: 41); OsIPT10 (SEQ ID NO: 59); OsIPT11 (SEQ ID NO: 43); ); OsIPT3 (SEQ ID NO: 63); OsIPT2 (SEQ ID NO: 46); OsIPT1 (SEQ ID NO: 49); OsIPT5 (SEQ ID NO: 52); OsIPT4 (34394150) (SEQ ID NO: 66); OsIPT7 (SEQ ID NO: 54); AtIPT1 (AB062607) (SEQ ID NO: 29); AtIPT3 (AB062610) (SEQ ID NO: 34); AtIPT4 (AB062611) (SEQ ID NO: 30); AtIPT5 (AB062608) (SEQ ID NO: 35); AtIPT6 (AB062612) (SEQ ID NO: 36); AtIPT7 (AB062613) (SEQ ID NO: 37); AtIPT8 (AB062614) (SEQ ID NO: 38); Sho (Petunia) (SEQ ID NO: 31 ) and consensus sequence (SEQ ID NO: 67).

Fig. 11 is a Northern blot showing the relative expression of ZmlPT2 gene in different parts of the grain at different days after pollination. Transcript levels of 0 to 25-DAP lobed grains were determined and quantified relative to the abundance of 18S RNA transcripts.

Figure 12 shows chromatograms of DMAPP::AMP prenyltransferase activity of recombinant proteins purified from Agrobacterium and maize.

Figure 13 shows the chromatogram of the reaction product in Figure 12 after further processing.

Figure 14 shows the chromatogram of DMAPP::ATP prenyltransferase activity of purified recombinant protein from corn.

Figure 15 is a Western blot of whole corn kernels at various days post pollination.

Figure 16 is a graphical representation of TUSC results.

Figure 17 is a phylogenetic tree of plant IPT sequences.

Detailed description of the invention

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings, which show some but not all technical solutions of the present invention. In fact, these inventions may be expressed in many different forms and should not be construed as limited to the technical solutions set forth herein; these technical solutions are provided so that the present invention will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

The various modifications and other technical solutions of the present invention listed here remind those skilled in the art that the present invention enjoys the benefit of the teachings shown in the above description and the associated drawings. Therefore, it should be understood that the present invention is not limited by specific technical solutions, and modifications and other technical solutions are also included in the scope of appended claims. Although specific terms are used herein, they are used in a generic, descriptive sense only and not for purposes of limitation.

combination

Compositions of the invention include isopentenyltransferase (IPT) polypeptides and polynucleotides involved in the regulation of plant development, morphology, and physiological processes. The compositions of the present invention further include an IPT promoter capable of regulating transcription. Particularly, the present invention provides isolated comprising the coding SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, Polynucleotides having amino acid sequences shown in 66 and 77 as nucleotide sequences. There is further provided a polynucleotide having as shown herein, for example, the sequence numbers are SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62, 64, 45, 48, 51, 56, 65, 69, 70, 71, 72, 73, An isolated polypeptide of the amino acid sequence encoded by 74 or 77. Another composition includes the IPT promoter sequence whose sequence number is SEQ ID NO: 25 or 75, and further provided herein are ZmlPT4 (SEQ ID NO: 5), ZmlPT5 (SEQ ID NO: 8) isolated and identified from the 5' region. ), ZmIPT6 (SEQ ID NO: 11), ZmIPT7 (SEQ ID NO: 14), ZmIPT8 (SEQ ID NO: 17), ZmIPT9 (SEQ ID NO: 20), OsIPT1 (SEQ ID NO: 47), OsIPT2 (SEQ ID NO: 44), OsIPT3 (SEQ ID NO: 62), OsIPT4 (SEQ ID NO: 64), OsIPT5 (SEQ ID NO: 50), OsIPT6 (SEQ ID NO: 55), OsIPT7 (SEQ ID NO: 53), OsIPT8 (SEQ ID NO: 40), OsIPT9 (SEQ ID NO: 60), OsIPT10 (SEQ ID NO: 58) and OsIPT11 (SEQ ID NO: 42) promoter sequences.

The prenyltransferase polypeptides of the invention have sequence identity to members of the prenyltransferase protein family. Polypeptides in the IPT family have been identified in various bacteria and in Arabidopsis and Petunia. See, eg, (Kakimoto (2001) Plant Cell Physio. 42:677-658); Takei et al. (2001) The Journal of Biological Chemistry 276:26405-26410 and Zubko et al. (2002) The Plant Journal 29:797 -808). IPT family members are characterized by the consensus sequence GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST] (SEQ ID NO: 32) (where x Indicates any amino acid residue, [] indicates that it can be any amino acid shown in [], x{m, n} indicates that there are m to n amino acids). See Kakimoto et al. (2001) Plant Cell Physiol. 42:677-85 and Kakimoto et al. (2003) J, Plant Res. 116:233-9; both incorporated herein by reference. IPT family members also have ATP/GTP binding sites. The amino acid sequence alignment of maize IPT protein and Arabidopsis and petunia cytokinin biosynthetic enzymes is shown in Figure 1, and the amino acid sequence alignment of rice IPT protein and Arabidopsis and petunia biosynthetic enzymes is shown in Figure 10 shown. Asterisks indicate consensus sequences found in multiple cytokinin biosynthetic enzymes. Underlined amino acids indicate putative ATP/GTP binding domains.

et al.(2000)Proc Natl Acad Sci 97：14778-14783和Takei et al.(2003)JPlant Res.116(3)：265-9。Prenyltransferases are involved in cytokinin biosynthesis, and thus the IPT polypeptides of the invention have "cytokinin synthesis activity". "Cytokinin synthetic activity" refers to an enzymatic activity that produces cytokinins and their derivatives or intermediates in any of the cytokinin synthetic pathways. Thus cytokinin synthesis activities include, but are not limited to, DMAPP:AMP isopentenyltransferase activity (conversion of AMP (adenosine-5'-monophosphate) and DMAPP to iPMP (isopentenyladenosine-5 '-monophosphate)), DMAPP:ADP isopentenyltransferase activity (converts ADP(adenosine-5'-diphosphate) and DMAPP to iPDP(isopentenyladenosine-5'-diphosphate)) , DMAPP: ATP isopentenyl transferase activity (converts ATP (adenosine-5'-triphosphate) and DMAPP into iPTP (isopentenyl adenosine-5'-triphosphate)) and DMAPP: tRNA isopentenyl Base transferase activity (modification of tRNAs in the cytoplasm and/or mitochondria to give prenyl groups). Cytokinin synthesis activity may further include substrates containing secondary side chain precursors in addition to DMAPP. Examples of side chain donors include compounds derived from terpenoids. For example, the substrate may be hydroxymethylbutenyl diphosphate (HMBPP), which allows for the synthesis of trans-zeatin nucleoside monophosphate (ZMP). See for example

et al. (2000) Proc Natl Acad Sci 97:14778-14783 and Takei et al. (2003) J Plant Res. 116(3):265-9.

Cytokinin synthesis activity further includes the synthesis of intermediates involved in ZMP formation. For example, in Takei et al. (2001) The Journal of Biological Chemistry 276: 26405-26410; Zubo et al. (2002) The Plant Journal 29: 797-808; Kakimoto et al. (2001) Plant Cell Physio. 42: 677 - 658 and Sun et al. (2003) Plant Physiology 131: 167-176, each of which is incorporated herein by reference, can be found in assays for the production of various cytokinins and their intermediates. "Cytokinin synthetic activity" also includes any phenotypic change in a plant or plant cell that is characterized by an increased concentration of a cytokinin. Such cytokinin-specific effects are discussed elsewhere herein and include, but are not limited to: enhanced shoot formation, reduced apical dominance, delayed senescence, delayed flowering, increased leaf growth, increased cytokinin levels in plants, increased Stress tolerance, reduced top grain abortion, increased or maintained seed fixation and reduced root growth under stress conditions. Methods for determining or detecting these phenotypes are known. See, eg, Miyawaki et al. (2004) The Plant Journal 37:128-138, Takei et al. (2001) The Journal of Biological Chemistry 276:26405-26410, Zubo et al. (2002) The Plant Journal 29:797-808 ; Kakimoto et al. (2001) Plant Cell Physio. 42: 677-658 and Sun et al. (2003) Plant Physiology 131: 167-176, each of which is incorporated herein by reference. Other phenotypes resulting from increased cytokinin synthesis activity in plants are also discussed herein.

The compositions of the invention include IPT sequences involved in cytokinin biosynthesis. Particularly, the present invention provides isolated comprising coding SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63 , 66 and 77 shown in the amino acid sequence of the polynucleotide sequence of the nucleotide sequence. It is further provided that the sequence number shown herein is SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62, 64, 45, 48, 51, 56, 65, 69, 70, 71, 72, 73 or 74 and fragments thereof Isolated polypeptides of the amino acid sequence encoded by the polynucleotides of the variants. In addition, promoter sequences such as SEQ ID NO: 25 or 75 and variants and fragments thereof are further provided.

The invention includes isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or substantially free from components with which it naturally accompanies, or from polynucleotides or proteins with which it interacts in its natural environment protein component. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques; and substantially free of chemical precursors or other compounds when chemically synthesized. Preferably, an "isolated" polynucleotide is one that is free of sequences (preferably protein coding sequence). For example, in various embodiments, the isolated polynucleotide comprises less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of the polynucleotide naturally flanking the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Nucleotide sequence. Proteins that are substantially free of cellular material include protein preparations that contain less than 30%, 20%, 10%, 5%, or 1% (dry weight) of contaminating protein. When the protein of the invention, or a biologically active portion thereof, is recombinantly produced, preferably the culture medium contains less than about 30%, 20%, 10%, 5% or 1% (dry weight) of chemical precursors or non-target proteins compound.

Fragments and variants of the inventive polynucleotides and their encoded proteins are also included in the present invention. "Fragment" refers to a portion of a polynucleotide or amino acid sequence and a portion of the protein it encodes. A polynucleotide fragment may encode a protein fragment that retains the biological activity of the native protein, thereby possessing cytokinin synthesis activity. Alternatively, polynucleotide fragments used as hybridization probes generally do not encode fragment proteins that retain biological activity. Accordingly, a nucleotide sequence fragment ranges from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides up to a full-length polynucleotide encoding a protein of the present invention.

An IPT polynucleotide fragment encoding a biologically active portion of an IPT protein of the invention encodes at least 15, 25, 30, 50, 100, 150, 200, 225, 250, 275, 300, 310, 315 or 320 consecutive amino acids, or All amino acids of the full-length IPT protein of the present invention (such as SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63 322, 364, 337, 338, 352, 388, 353, 352, 450, 590, 328, 325, 251, 427, 417, 585, 455, 344 and 347 amino acids in and 66). IPT polynucleotide fragments that serve as hybridization probes or PCR primers generally need not encode a biologically active portion of an IPT protein.

Accordingly, an IPT polynucleotide fragment may encode a biologically active portion of an IPT protein, or may be a fragment used as a hybridization probe or PCR primer using the methods described below. The biologically active portion of the IPT protein can be prepared by isolating an IPT polynucleotide fragment expressing the encoded IPT protein portion of the present invention (for example, by recombinant expression in vitro), and measuring the activity of the IPT protein encoding portion. A polynucleotide that is a fragment of an IPT nucleotide sequence comprising at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900 , 950 or 965 contiguous nucleotides, or up to the number of nucleotides of the full-length IPT polynucleotide described herein (e.g., SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62, 64, 45, 48, 51, 1495, 969, 2901, 2654, 1095, 4595, 1014, 1955, 1017, 1652, 1059, 3419, 1167, 1535, 3000, 1209, 1062, 56, 65, 69, 70, 71, 72, 73, and 74 1299, 1056, 4682, 8463, 4470, 4114, 2599, 1284, 5030, 8306, 7608, 5075, 4777, 984, 975, 753, 1254, 1044, 1035, 1284, 1353, 1368, 1758, and 1773 nucleosides acid).

"Variants" refer to substantially identical sequences. For polynucleotides, a variant is one or more positions in the natural polynucleotide comprising a deletion and/or addition of one or more nucleotides, and/or one or more positions in the natural polynucleotide There are one or more nucleotide substitutions. As used herein, "native" polynucleotides and polypeptides contain a native nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of an IPT polypeptide of the present invention due to the degeneracy of the genetic code. Such natural variants can be identified by known molecular biology techniques, such as the polymerase chain reaction (PCR) and the hybridization techniques described below. Variant polynucleotides also include synthetically derived polynucleotides, such as sequences prepared by site-directed mutagenesis that still encode an IPT protein of the present invention. Generally, variants of a particular polynucleotide of the invention have at least 40%, 45%, 50%, 55%, 60%, 65% identity with a particular polynucleotide as determined elsewhere herein by sequence alignment programs and parameters. %, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity .

Variants of a particular polynucleotide of the invention (eg, a reference polynucleotide) can be assessed by comparing the percent sequence similarity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, also described encoding with SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 An isolated polynucleotide having a certain percentage sequence identity to the polypeptide of or 77 . The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. For any given pair of polypeptides of the invention, when assessed by comparing the percent sequence identities of the two encoded polypeptides, the percent sequence identity between the two encoded polypeptides is at least 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or higher sequence identity.

A "variant" protein is one derived from a native protein by deletion or addition of one or more amino acids at one or more positions in the native protein, and/or one or more amino acid substitutions at one or more positions in the native protein come protein. The specific variant proteins included in the present invention are biologically active, that is to say, they have the expected biological activity of the native protein, ie the cytokinin synthesis activity described herein. Such variants can be obtained by, for example, genetic polymorphism or human manipulation. The biologically active variants of the native IPT protein of the present invention have at least 40%, 45%, 50%, 55%, 60%, 65% of the amino acid sequence of the native protein as determined by sequence alignment programs and parameters described elsewhere herein. , 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity. Biologically active variants of a protein of the invention may differ from the protein by 1-15, 1-10, eg 6-10, 5, 4, 3, 2 or even 1 amino acid residue.

The proteins of the invention can be altered in a variety of ways including amino acid substitutions, deletions, cleavages and insertions. These manipulations are generally known in the art. For example, amino acid sequence variants and fragments of IPT proteins can be prepared by DNA mutation. Methods of mutation and polynucleotide alteration are known in the art. See, e.g., Kunkel (1985) Proc.Natl.Acad.Sci.USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol.154:367-382; ) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model in Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereby incorporated by reference integrated into the text. Conservative substitutions, such as exchanging an amino acid with another with the same properties, are most desirable.

Thus, the genes and polynucleotides of the invention include both native sequences and mutated forms. Likewise, the proteins of the present invention include native proteins and variants and modified forms thereof. This variant still has the expected IPT activity. Clearly, mutations in the DNA encoding the variant will not place the sequence out of reading frame and, preferably, will not create complementary regions that would produce secondary mRNA structure.

Deletions, insertions and substitutions of the proteins included herein are not expected to result in fundamental changes in the properties of the proteins. However, when it is difficult to predict the precise effects of substitutions, deletions or insertions in advance, those skilled in the art can predict these effects through conventional screening methods. That is, the activity of cytokinin synthesis was estimated by measuring its activity. See, e.g., Takei et al. (2001) The Journal of Biological Chemistry 276:26405-26410; Zubo et al. (2002) The Plant Journal 29:797-808; Kakimoto et al. (2001) PlantCell Physio.42:677- 658; Sun et al. (2003) Plant Physiology 131: 167-176 and Miyawaki et al. (2004) The Plant Journal 37: 128-138. All references are incorporated into the text by reference.

Variant polynucleotides and proteins also encompass sequences and proteins derived from mutational and recombinant techniques such as DNA shuffling. By such techniques, one or more different IPT coding sequences can be manipulated to obtain new IPT polypeptides with desired properties. Likewise, a library of recombinant polynucleotides can be generated from a population of related sequence polynucleotides containing sequence regions of substantially identical sequence, and subjected to homologous recombination in vitro or in vivo. For example, using this approach, sequence elements encoding domains of interest can be recombined between the IPT gene of the invention and other known IPT genes, resulting in new genes encoding proteins with improved properties of interest - such as increased _K of the enzyme . Such DNA shuffling strategies are known in the art. See, e.g., Stemmer (1994) Proc. et al. (1997) J. Mol. Biol. 272: 336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri et al. (1998) Nature 391: 288-291 and US Patents 5,605,793 and 5,837,458.

Compositions of the present invention also include the isolated polynucleotide that comprises the IPT promoter nucleotide sequence listed in SEQ ID NO: 25 or 75, and be further separated, by 5' region to as SEQ ID NO: 5 (ZmIPT4), SEQ ID NO: 8 (ZmIPT5), SEQ ID NO: 11 (ZmIPT6), SEQ ID NO: 14 (ZmIPT7), SEQ ID NO: 17 (ZmIPT8), SEQ ID NO: 20 (ZmIPT9), SEQ ID NO: 20 (ZmIPT9), SEQ ID NO: ID NO: 47 (OsIPT1), SEQ ID NO: 44 (OsIPT2), SEQ ID NO: 62 (OsIPT3), SEQ ID NO: 64 (OsIPT4), SEQ ID NO: 50 (OsIPT5), SEQ ID NO: 55 ( OsIPT6), SEQ ID NO: 53 (OsIPT7), SEQ ID NO: 40 (OsIPT8), SEQ ID NO: 60 (OsIPT9), SEQ ID NO: 58 (OsIPT10) and a portion of SEQ ID NO: 42 (OsIPT11) A characteristic promoter sequence providing the coding sequence.

"Promoter" refers to a regulatory region of DNA, usually including a TATA box that directs RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site of a specific polynucleotide sequence. A promoter may additionally include other recognition sequences, usually upstream or 5' of the TATA box, referred to as upstream promoter elements, which may affect the rate of transcription initiation. The promoter sequences of the present invention can regulate (ie repress or activate) transcription.

It is known that additional domains may be added to the promoter sequences of the invention to regulate the level of expression, the timing of expression or the type of tissue in which expression occurs. In particular, see Australian Patent AU-A-77751/94 and US Patents 5,466,785 and 5,635,618.

The present invention also includes fragments and variants of the inventive IPT promoter polynucleotides. Fragments of the promoter polynucleotide can maintain biological activity and thus have transcriptional regulatory activity. Alternatively, the polynucleotide fragments used as hybridization probes are biologically inactive. Accordingly, promoter nucleotide sequence fragments can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to the full length of a polynucleotide of the invention.

Accordingly, IPT promoter polynucleotide fragments may encode biologically active portions of the IPT promoter, or be fragments useful as hybridization probes or PCR primers in the methods described below. The biologically active portion of an IPT promoter polynucleotide can be obtained by isolating a portion of an IPT promoter polynucleotide of the present invention and measuring the activity of the IPT promoter portion. Polynucleotides that are IPT promoter fragments comprising at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000 , 1,050 or 1,080 contiguous nucleotides, or the number of nucleotides of the full-length IPT promoter polynucleotide of the present invention (eg, 1082 and 1920 nucleotides in SEQ ID NOS: 25 and 75, respectively).

For promoter polynucleotides, variants include deletions and/or additions of one or more nucleotides at one or more internal sites in the native polynucleotide, and/or at one or more A position has one or more nucleotide substitutions. Typically, variants of a particular promoter polynucleotide of the invention will have a sequence identity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

Variant polynucleotides also include sequences derived from mutational and recombinant techniques such as DNA shuffling. By this technique, one or more different promoter sequences can be manipulated to obtain an IPT promoter with the desired properties. Strategies for such DNA shuffling are described elsewhere herein.

Methods are known in the art to determine whether a promoter sequence still retains the ability to regulate transcription. This activity can be determined by Northern blot analysis. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), incorporated herein by reference. Alternatively, the biological activity of a promoter can be determined by methods designed to measure the activity and/or level of polypeptide expressed from the promoter. These assay methods are known in the art. Furthermore, known promoter elements can be identified within the putative promoter sequence. For example, the IPT1 promoter of the invention (SEQ ID NO: 25) has a TATA box at bp688. A TATA box-like sequence can be found 48 bp upstream of the transcription start site (between bp 1035 and 1042). A possible CAAT box can be found between bp 929 and 932.

The polynucleotides (ie IPT sequences and IPT promoter sequences) of the present invention can be used to isolate corresponding sequences from other organisms, especially other plants, especially other monocotyledonous plants. In this manner, the sequences can be identified based on their sequence homology to the sequences listed herein by methods such as PCR, hybridization and other methods. The present invention includes sequences isolated on the basis of sequence identity to the complete IPT sequence or IPT promoter sequence set forth herein, or variants and fragments thereof. Such sequences include sequences orthologous to the inventive sequences. "Orthologs" refer to genes derived from a common ancestral gene that exist in different species as a result of speciation. Genes found in different species have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% of their nucleotide sequence and/or their encoded protein sequence , 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity are considered to be orthologs. The functions of orthologs are often highly conserved across species. Accordingly, the present invention includes isolated polynucleotides encoding an IPT protein or comprising an IPT promoter sequence that hybridize under stringent conditions to an IPT sequence or IPT promoter sequence described herein, or a variant or fragment or complement thereof

In the PCR method, oligonucleotide primers can be designed for PCR reactions to amplify the corresponding DNA sequences from cDNA or genomic DNA extracted from any target plant. Methods for designing PCR primers and PCR cloning are known in the art, see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCRStrategies (Academic Press, New York) and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.

In hybridization techniques, all or part of a known polynucleotide can be used as a means of selectively hybridizing to other corresponding polynucleotides present in cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) of the organism of choice. probe. Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides that can be labeled with a detectable moiety such ^as32P or other detectable label. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on the IPT polynucleotide or IPT promoter sequence of the present invention. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are known in the art, see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York) .

For example, entire IPT polynucleotides or IPT promoter sequences described herein, or one or more portions thereof, can be used as probes that specifically hybridize to corresponding IPT polynucleotides, messenger RNAs or promoter sequences. In order to obtain specific hybridization under various conditions, these probes include specific sequences in the IPT polynucleotide sequence or the IPT promoter sequence, and are preferably at least about 10 nucleotides in length, more preferably at least about 20 nucleotides in length. nucleotides. Such probes can be used to amplify the corresponding IPT polynucleotide or IPT promoter from selected plants by PCR. This technique can be used to isolate additional coding sequences from desired plants, or as a diagnostic method to determine the presence of coding sequences in plants. Hybridization techniques include hybridization screening of DNA libraries on plates (either as plaques or clones; see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Hybridization of these sequences is performed under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" refer to conditions under which a probe hybridizes to its target sequence substantially higher than to other sequences (eg, at least 2-fold higher than background). Stringent conditions are sequence-dependent and vary in different circumstances. By controlling hybridization stringency and/or wash conditions, target sequences that are 100% complementary to the probe (homologous probes) can be identified. Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequences, so that lower degrees of similarity (heterologous probes) can be detected. Typically, probes are less than about 1000 nucleotides in length, preferably less than about 500 nucleotides in length.

Typically, stringent conditions refer to pH 7.0 to 8.3 and a temperature of at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). When the salt concentration is less than 1.5M Na ion, typically 0.01 to 1.0M Na sodium ion concentration (or other salt) conditions. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. Recommended low stringency conditions include hybridization at 37°C in a buffer containing 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecylsulfonate) in 1X to 2X SSC (20X SSC =3.0M NaCl/0.3M sodium citrate), rinse at 50 to 55°C. Recommended conditions of moderate stringency include hybridization in 40 to 45% formamide, 1M NaCl, 1% SDS at 37°C and washes in 0.5X to 1X SSC at 55 to 60°C. Recommended highly stringent conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C and washes in 0.1X SSC at 60 to 65°C. Additionally, the wash buffer may contain about 0.1% to 1% SDS. The hybridization time is generally less than 24 hours, usually about 4 to 12 hours. The rinse time should be at least long enough to achieve equilibrium.

Specificity is mainly a function of post-hybridization rinsing, and the key factors are the ionic strength and temperature of the final rinsing solution. For DNA-DNA hybrids, T _m can be roughly calculated by the equation of Meinkothand Wahl (1984) Anal.Biochem.138: 267-284: T _m = 81.5 ° C + 16.6 (log M) + 0.41 (% GC) -0.61(%form)-500/L; where M is the number of moles of monovalent cations, %GC number is the percentage of guanine and cytosine in DNA, %form is the percentage of formamide in the hybridization solution, and L is the hybrid base base pair length. The _Tm refers to the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. _Tm decreases by about 1°C for every 1% mismatch; thus _Tm , hybridization and/or wash conditions can be adjusted to hybridize to sequences of desired identity. For example, if one is looking for sequences > 90% identical, the _Tm can be lowered by 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point ( _Tm ) for the specific sequence and its complement at a defined ionic strength and pH. However, very stringent conditions at 1, 2, 3, or 4°C lower than the thermal melting point ( _Tm ) can also be used for hybridization and/or washing; moderately stringent conditions at 6°C lower than the thermal melting point ( _Tm ) , 7, 8, 9, or 10°C for hybridization and/or washing; low stringency conditions for hybridization at 11., 12, 13, 14, 15, or 20°C lower than the thermal melting point (T _m ) and/or rinse. By equation, hybridization and wash components and desired _Tm , those skilled in the art will understand that variations in the stringency of hybridization and/or wash conditions are also described. If the desired degree of mismatching results in a _Tm below 45°C (aqueous solution) or 32°C (formamide solution), the SSC concentration is preferably increased so that higher temperatures can be used. The detailed guidance of nucleic acid hybridization is referring to Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part 1, Chapter 2 (Elsevier, New York) and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

The following terms are used to describe the relationship between two or more polynucleotides or polypeptides: (a) "reference sequence" (b) "comparison window" (c) "sequence identity" and (d) "sequence identity" percentage".

(a) "Reference sequence" as used herein refers to a defined sequence that is the basis for sequence comparison. A reference sequence may be part or all of a specific sequence; for example as part of a full-length cDNA or gene sequence or as a complete cDNA or gene sequence.

(b) "Comparison window" as used herein refers to a contiguous and specific portion of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window is compared to a reference sequence (excluding additions or deletions), which may contain additions or deletions ( gaps) for optimal alignment of the two polynucleotides. Typically, the comparison window is at least 20 contiguous nucleotides in length, preferably 30, 40, 50, 100 or longer. It will be appreciated by those skilled in the art that to avoid high similarity to a reference sequence due to the presence of gaps in a polynucleotide sequence, a gap penalty should be introduced and subtracted from the number of matches.

Methods of alignment of sequences for comparison are known in the art. Thus, the percent sequence identity between any two sequences can be determined by a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv.Appl.Math.2:482; Needleman and Wunsch (1970) J.Mol.Biol.48:443-453 global alignment algorithm; Pearson and Lipman (1988) Proc.Natl.Acad.Sci.85:2444-2448 local search alignment algorithm; Karlin and Altschul (1990 ) Proc.Natl.Acad.SciUSA 872264 algorithm, modified from Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877.

These computer-implemented programs of mathematical algorithms allow the comparison of sequences to determine sequence identity. Such executive programs include, but are not limited to, CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (version 2.0) and GAP, BESTFIT, BLAST in version 10 of the GCG Wisconsin Genetics software package , FASTA and TFASTA (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments are performed by these programs using default parameters. CLUSTAL program in Higgins et al. (1988) Gene73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang It is well described in et al. (1992) CABIOS 8: 155-65 and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) described above. When comparing amino acid sequences with the ALIGN program, a PAM120 weighted residue table, a gap length penalty of 12 and a single gap penalty of 4 can be used. The BLAST program of Altschul et al (1990) J. Mol. Biol. 215:403 is based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to proteins or polypeptides of the invention. To obtain gapped alignments for comparison, Gapped BLAST (in BLAST 2.0) as described by Altschul et al. (1997) Nucleic Acids Res. 25:3389 can be used. Alternatively, PSI-BLAST (BLAST 2.0) can be used to perform iterative searches to detect distant relationships between molecules. See Altschul et al. (1997), supra. When using BLAST, GappedBLAST, PSI-BLAST, the default parameters of the respective programs (eg, BLASTN for nucleotide sequences, BLASTX for protein sequences) can be used. See www.ncbi.nlm.nih.gov . Alignment can also be performed by manual inspection.

Unless otherwise stated, sequence identity/similarity values provided herein refer to those obtained using GAP version 10 according to the following parameters: % identity and % similarity for nucleotide sequences with a GAP weight of 50 and a length weight of 3, The nwsgapdna.cmp scoring matrix was used; for % identity and % similarity of amino acid sequences, with a GAP weight of 8 and a length weight of 2, the BLOSUM62 scoring matrix was used; or any equivalent program thereof. "Equivalent program" means any alignment that yields, for any two sequences under study, an alignment that has identical nucleotide or amino acid residue matches and has the same percentage when compared to the corresponding alignment produced by GAP version 10 A sequence comparison program for sequence identity.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453 to find the alignment of two complete sequences with the largest number of matches and the smallest number of gaps. GAP considers all possible alignments and gap positions and obtains the alignment with the largest number of matching bases and the fewest gaps. It takes into account a gap creation penalty and a gap extension penalty in matching base units. GAP must make it advantageous to generate a penalty number for each matching gap obtained by inserting a gap. Additionally, if the Gap Extension Penalty is chosen to be greater than 0, GAP must multiply the Gap Length of each inserted gap by the Gap Extension Penalty to be favorable. In the GCG Wisconsin Genetics package version 10, the default gap creation penalty and gap extension penalty are 8 and 2 for protein sequences, respectively. For nucleotide sequences, the default gap creation penalty is 50, and the default gap extension penalty is 3. Gap creation and gap extension penalties are expressed as integers selected from 0 to 200, optional. Thus, for example, gap creation and gap extension penalties could be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or larger.

GAP is a member of the family of the best sequence alignments. There are many members in this family, but no other is of better quality. GAP shows four advantages in sequence alignment: quality, ratio, identity and similarity. Mass is the largest unit when aligning sequences. The ratio is the mass divided by the number of bases of the shorter fragment. Percent identity is the percentage of symbols that actually match. Percent similarity is the percentage of similar symbols. Signs corresponding to gaps are ignored. When the scoring matrix of a pair of symbols is greater than or equal to the similarity threshold of 0.50, the similarity is scored. The scoring matrix used in the GCG Wisconsin Genetics software package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

(c) "Sequence identity" or "identity" of two polynucleotide or polypeptide sequences herein refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When the percent sequence identity refers to a protein, residue positions that are considered to be inconsistent usually differ by conservative amino acid substitutions, in which an amino acid residue is replaced by another amino acid residue having the same property, such as charge or hydrophobicity, Thereby without changing the functional properties of the molecule. When sequences differ at conservative substitutions, the percent sequence identity can be adjusted to correct for the conservative nature of these substitutions. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Methods for making such adjustments are known to those skilled in the art. Typically, conservative substitutions are included to score as partial rather than full mismatches, thereby increasing the percent sequence identity. Thus, for example, conservative substitutions are scored between 0 and 1 when identical amino acids are scored as 1 and non-conservative substitutions are scored as 0. Scores for conservative substitutions can be calculated, for example, by the PC/GENE program (Intelligenetics, Mountain View, California).

(d) "Percent sequence identity" as used herein refers to the value obtained by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence of the comparison window is identical to a reference sequence (excluding additions or deletions). ) comparisons may include additions or deletions (such as gaps) to optimize the alignment of the two sequences. The percentage is calculated by determining the number of identical nucleotide base or amino acid residue positions in the two sequences to obtain the number of matching positions, dividing the total number of comparison window positions by the number of matching positions, and multiplying the result by 100 to obtain the percent sequence identity.

The invention further provides plants comprising an altered level and/or activity of an IPT polypeptide of the invention. In some technical solutions, the plant of the invention has the IPT sequence of the invention stably integrated in its genome. In other embodiments, plants genetically altered at the loci encoding the IPT polypeptides of the invention are provided. "Native locus" refers to a naturally occurring gene sequence. For certain embodiments, the locus is listed in SEQ ID NO: 21, 40, 42, 44, 47, 50, 53, 55, 58, 60, 62 or 64. Genetic loci can be altered to reduce or eliminate the activity of the IPT polypeptide. The term "genetic alteration" as used herein refers to the alteration of the genetic information of a plant or plant part by the insertion of one or more exogenous polynucleotides, which can result in a phenotypic change in the plant. By "phenotypic change" is meant a detectable change in one or more cellular functions. For example, a plant having a genetic alteration at the locus encoding an IPT polypeptide can reduce or eliminate the expression or activity of the IPT polypeptide. Various methods for generating such genetically altered loci, and resulting in various phenotypic changes due to altered levels/activity of the IPT sequences of the invention are described elsewhere herein.

The invention further provides plants having at least one DNA construct comprising a desired heterologous nucleotide sequence linked to an IPT promoter of the invention. In a further embodiment, the DNA construct is stably integrated in the plant genome.

The term plant as used herein includes whole plants, plant parts or organs (eg leaves, stems, roots), plant cells and seeds, and progeny thereof. Plant cells as used herein include, but are not limited to, cells obtained from seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores, and plant protoplasts and plant cell tissue cultures, plant calli, plant clumps, and complete plant cells of plants and plant parts—such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, ears, ears of corn , shell, stalk, root, root tip, anther, grain, etc. "Grain" as used herein refers to the mature seed produced for commercial cultivation purposes other than the purpose of growing or multiplying the species. Progeny, variants and mutants of regenerated plants are also included within the scope of the present invention as long as they contain the introduced nucleic acid sequence.

method

I. Provide sequence

The sequences of the invention can be introduced/expressed in host cells such as bacteria, yeast, insects, mammals or preferably plant cells. It should be appreciated that those skilled in the art are familiar with a variety of systems by which a polypeptide or nucleotide sequence of the invention can be introduced into a host cell. The various known methods of providing proteins in prokaryotes or eukaryotes are therefore not described in detail.

"Host cell" refers to a cell containing a heterologous nucleic acid sequence of the present invention. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. The host cell can also be a monocot or a dicot cell. In a particular embodiment, the monocot host cell is a maize host cell.

The use of the term "polynucleotide" does not limit the invention to DNA-containing polynucleotides. Those skilled in the art know that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include natural molecules and synthetic analogs. The polynucleotides of the invention also encompass all sequence forms including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like.

The IPT polynucleotide or IPT promoter of the present invention may be provided in an expression cassette for expression in a plant of interest. The expression cassette includes 5' and 3' regulatory sequences operably linked to the IPT polynucleotides of the invention. "Operably linked" refers to the functional linkage of two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (ie, a promoter) is a functional linkage that allows expression of the polynucleotide of interest. Operably linked elements may be continuous or discontinuous. When referring to the joining of two protein coding regions, operably linked means that the coding regions are in the same reading frame. The expression cassette may additionally contain at least one other gene to be co-transformed into the organism. Alternatively, other genes may be provided in multiple expression cassettes. The expression cassette can provide multiple restriction sites and/or recombination sites for insertion of the IPT polynucleotide so that it is under the transcriptional control of the regulatory regions. The expression cassette may additionally contain a selectable marker gene.

In a specific technical solution, the expression cassette includes a 5'-3' direction of transcription functioning in plants, a transcriptional and translational initiation region (i.e. a promoter), an IPT polynucleotide of the present invention, and a transcriptional and translational termination region (i.e. end zone). Regulatory regions (ie promoters, transcriptional regulatory regions and translational termination regions) and/or IPT polynucleotides of the invention may be native/similar to the host cell or to each other. Alternatively, the regulatory regions and/or the IPT polynucleotides of the invention may be heterologous to the host cell or to each other. "Heterologous" as used herein means that its sequence is derived from a foreign species, or, if from the same species, components and/or genetic loci of the native form have been substantially altered by deliberate human intervention. For example, the promoter operably linked to the heterologous polynucleotide is from a species different from the source of the polynucleotide, or, if from the same/similar species, one or both of which occur in their original form and/or locus have been substantially altered, or the promoter is not the native promoter of the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence.

When a heterologous promoter can be used to express the IPT sequence, the native promoter sequence or other IPT promoters (such as SEQ ID NO: 25 or 75) can be used. Such constructs alter the expression level of IPT sequences in plants or plant cells. Thus, the phenotype of a plant or plant cell can be altered.

The termination region may be native to the transcriptional initiation region, may be native to the operably linked target IPT polynucleotide, may be native to the plant host, or may be derived from other sources (e.g., foreign or heterologous to the reference promoter). source), a target IPT polynucleotide, a plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of Agrobacterium tumefaciens, such as the carboxyethylarginine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903 and Joshi et al. (1987) Nucleic Acids Res. 15: 9627-9639.

Where appropriate, polynucleotides may be optimized for increased expression in transformed plants. That is, polynucleotides can be synthesized using plant-preferred codons to increase expression. See, eg, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. These methods are available in the field of synthetic plant preference genes. See, eg, US Patents 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, incorporated herein by reference.

Other sequence changes in the cellular host are known to enhance gene expression. These include deletions encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such characteristic sequences that may be detrimental to gene expression. For a given cellular host, the G-C content in the sequence can be adjusted to the average level calculated by reference to the known expressed genes in the host cell. Where possible, sequence changes were made to avoid possible hairpin secondary mRNA structures.

The expression cassette may additionally contain a 5' leader sequence. Such leader sequences enhance translation. Translation leaders are known in the art and include picornavirus leaders, such as the EMCV leader (5' noncoding region of encephalomyocarditis) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); Potato virus Y leader region, for example TEV leader region (tobacco etch virus) (Gallie et al. (1995) Gene 165 (2): 233-238), MDMV leader region (maize dwarf mosaic virus ) (Virology 154:9-20), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); alfalfa mosaic virus capsid protein mRNA untranslated leader ( AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); Tobacco mosaic virus leader region (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, NewYork), pp.237-256); and the maize chlorotic mottle virus vector (MCMV) (Lomme et al. (1991) Virology 81:382-385). See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other known methods of enhancing translation can also be used.

In preparing expression cassettes, various DNA fragments can be manipulated to provide DNA sequences in the correct orientation and in the correct reading frame. To accomplish this, adapters or linkers can be used to join the DNA fragments, or other manipulations such as restriction sites, removal of spurious DNA, removal of restriction sites, etc., can be used as convenient. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution - such as transitions and transversions - can be used.

The expression cassette may also include a selectable marker gene for selection of transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes encoding resistance to antibiotics such as neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and to antibiotics such as glufosinate-ammonium, bromoxynil, juvenil and 2, Herbicide compounds such as butyl 4-dichlorophenoxyacetate (2,4-D) express resistance genes. Other selectable markers include such as β-galactosidase, fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85: 610-9 and Fetter et al. (2004) Plant Cell 16 : 215-28), cyan fluorescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117: 943-54 and Kato et al. (2002) Plant Physiol 129: 913-42), and yellow fluorescent protein (PhiYFP ^™ from Evrogen, see Bolte et al. (2004) J. Cell Science 117:943-54) such phenotypic markers. For other selectable markers, see generally Yarranton (1992) Curr.Opin.Biotech.3: 506-511; Christopherson et al. (1992) Proc. 1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA 86: 5400 -5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen (1993) Ph.D. dissertation, University of Heidelberg; Reines et al. al.(1993) Proc.Natl.Acad.Sci.USA 90:1917-1921; Labow et al.(1990) Mol.Cell.Biol.10:3343-3356; Zambretti et al.(1992) Proc.Natl. Acad.Sci.USA89:3952-3956; Baim et al.(1991) Proc.Natl.Acad.Sci.USA88:5072-5076; Wyborski et al.(1991) Nucleic Acids Res.19:4647-4653; Wissman (1989) Topics Mol.Struc.Biol.10:143-162; Degenkolb et al. (1991) Antimicrob.Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) PhD dissertation, Heidelberg University; Gossen et al. (1992) Proc.Natl.Acad.Sci.US A89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol.78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. These documents are incorporated herein by reference. The selectable marker genes listed above are not limiting. Any selectable marker gene can be used in the present invention.

A variety of promoters can be used in the practice of the present invention, including the promoter native to the polynucleotide sequence of interest. A promoter can be selected according to the desired outcome. Nucleic acids can be combined with constitutive, inducible, tissue-biased or other promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter in WO 99/43838 and U.S. Patent 6,072,050 and others; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810 -812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christense et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. ( 1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J.3: 2723-2730); ALS promoter (US Patent 5,659,026) and the like. Other constitutive promoters include, for example, US Patents 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;

Tissue-preferred promoters can be used to enhance IPT expression in specific plant tissues. Tissue-biased promoters include Yamamoto et al. (1997) Plant J.12 (2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38 (7): 792-803; Hansen et al. ( 1997) Mol.Gen Genet.254(3):337-343; Russell et al.(1997) Transgenic Res.6(2):157-168; Rinehart et al.(1996) Plant Physiol.112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascini et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam(1994) Results Probl.Cell Differ.20:181-196; Orozco et al.(1993) Plant Mol Biol.23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20): 9586-9590 and Guevara-Garcia et al. (1993) Plant J. 4(3): 495-505. Such promoters can be modified to attenuate expression, if desired. See also US Patent 2003/0074698, incorporated herein by reference.

Leaf-biased promoters are known in the art. See, eg, Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kwon et al. (1994) Plant Physiol. 105: 357-67; Yamamoto et al. (1994) Plant Cell Pnysiol. 35 (5): 773-778; Gotor et al. (1993) Plant J.3: 509-18; Orozco et al. (1993) Plant Mol. Biol.23 (6): 1129-1138; Baszczynski et al. ( 1988) Nucl.Acid Res.16: 4732; Mitra et al. (1994) Plant Molecular Biology 26: 35-93; Kayaya et al. (1995) Molecular and General Genetics 248: 668-674 and Matsuoka et al. (1993) Proc . Natl. Acad. Sci. USA 90(20): 9586-9590. Attenuation-regulated promoters can also be used, such as SAM22 (Crowell et al. (1992) Plant Mol. Biol. 18:459-466). See US Patent 5,589,052, incorporated herein by reference.

Root-biased promoters are known and can be selected from a number of existing literatures, or isolated de novo from a variety of alternative species. See, e.g., Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamate synthase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element of lentil GRP 1.8 gene); Sanger et al. (1990) Plant Mol.Biol.14 (3): 433-443 (Agrobacterium tumefaciens mannopine synthase (MAS) gene root-specific promoter) and Miao et al. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytoplasmic glycine synthase (GS), which can be expressed in soybean roots and root nodules). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, which describe two root-specific promoters for the hemoglobin gene isolated from the nitrogen-fixing non-legume Parasponia andersonii and the related non-nitrogen-fixing non-legume Trematomentosa. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into the non-legume Nicotiana tabacum and the legume Lotus corniculatus, both of which maintained root-specific promoter activity. Leach and Aoyagi (1991) described the promoters of the highly expressed rolC and rolD root-inducible genes in Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1):69-76). They suggest that enhancers and tissue-biased DNA determinants are dissociated in these promoters. Teeri et al. (1989) used the fusion with the lacZ gene to show that the T-DNA gene encoding octopine synthase in Agrobacterium is particularly active in the root apical epidermis, and that the TR2' gene is root-specific in intact plants and is absorbed by leaf tissues. wound irritation; together with insecticide or larvicide genes is a particularly desirable combination of useful features (see EMBO J.8(2):343-350). The fusion of the TR1' gene to nptII (neomycin phosphotransferase II) exhibited similar features. Other root-biased promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29 (4): 759-772); the rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691 and CRWAQ81 root-specific promoter with ADH first intron (U.S. Patent Publication 2005/0097633). See U.S. Patents 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,023,179.

A "seed-biased" promoter refers to a promoter that is active during seed development, and may include expression in early seed or related maternal tissues. Such seed-biased promoters include, but are not limited to, Cim1 (cytokinin-inducible messenger); cZ19B1 (maize 19 kDa zein); milps (inositol-1-phosphate synthase) (see WO 00/11177 and U.S. Patent 6,225,529, incorporated herein by reference). γ-zein is an endosperm-specific promoter. Glob-1 (Glob-1) is a typical embryo-specific promoter. For dicotyledonous plants, seed-specific promoters include, but are not limited to, soybean β-phaseolin, rapeseed protein, β-conglycinin, soybean lectin, cruciferin, and the like. For monocotyledonous plants, seed-specific promoters include, but are not limited to, maize 15kDa zein, 22kDa zein, 27kDa zein, γ-zein, wax gene, shrunken 1, shrunken 2, globulin 1, etc. See WO 00/12733, which describes seed-biased promoters from end1 and end2; incorporated herein by reference. In Sato et al. (1996) Proc. Natl. Acad. Sci. 93: 8117-8122; Nakase et al. (1997) Plant J 12: 235-46 and Postma-Haarsma et al. (1999) Plant Mol. Biol Other embryo-specific promoters are described in .39:257-71. In Albani et al. (1984) EMBO3:1405-15; Albani et al. (1999) Theor.Appl.Gen.98:1253-62; Albani et al. (1993) Plant J.4:343-55; Mena et al. Other endosperm-specific promoters are described in al. (1998) The Plant Journal 116: 53-62 and Wu et al. (1998) Plant Cell Physiology 39: 885-889.

Also of interest are promoters active in meristems such as developing inflorescence tissue, and promoters that drive expression during anthesis or early grain development. This can include, for example, the maize Zag promoter, including Zag1 and Zag2 (see Schmidt et al. (1993) The Plant Cell 5:729-37; GenBank X80206; Theissen et al. (1995) Gene 156:155-166 and U.S. Patent Application 10/817,483); maize Zap promoter (also known as ZmMADS; U.S. Patent Application 10/387,937; WO 03/078590); maize ckx1-2 promoter (U.S. Patent Application 2002-0152500 A1; WO02/0078438); maize eep1 Promoter (US patent application 10/817,483); maize end2 promoter (US patent 6,528,704 and US patent application 10/310,191); maize lecl promoter (US patent application 09/718,754); maize F3.7 promoter (Baszczynski et al ., Maydica 42: 189-201 (1997)); Maize tb1 promoter (Hubbarda et al., Genetics 162: 1927-1935 (2002) and Wang et al. (1999) Nature 398: 236-239); Maize eep2 promoter (U.S. Patent Application 10/817,483); Maize Thioredoxin H Promoter (U.S. Pending Patent Application 60/514,123); Maize Zm40 Promoter (U.S. Patent 6,403,862 and WO 01/2178); Maize mLIP15 Promoter (U.S. Patent 6,479,734); maize ESR promoter (U.S. Patent Application 10/786,679); maize PCNA2 promoter (U.S. Patent Application 10/388,359); maize cytokinin oxidase promoter (U.S. Patent Application 11/094,917); in Weigal et al .(1992) Cell69: 843-859; accession number AJ131822; accession number Z71981; promoter of accession number AF049870, and shoot bias described in McAvoy et al. (2003) Acta Hort. (ISHS) 625: 379-385 tropic promoter. In Ito et al. (1994) Plant Mol. Biol. 24: 863-878; Regad et al. (1995) Mo. Gen. Genet. 248: 703-711; Shaul et al. (1996) Proc. Natl. Acad. Sci. 93: 4868-4872; Ito et al. (1997) Plant J. 11: 983-992 and Trehin et al. (1997) Plant Mol. Meristem-biased promoters, all references are incorporated herein by reference.

Inflorescence-biased promoters include chalcone synthase promoter (Van der Meer et al. (1990) Plant Mol. Biol. 15:95-109), LAT52 (Twell et al. (1989) Mol. Gen. Genet .217:240-245), pollen specific gene (Albani et al (1990) Plant Mol Biol.15:605, Zm13 (Buerrero et al. (1993) Mol.Gen. Genet.224:161-168), maize pollen Specific genes (Hamilton et al. (1992) Plant Mol. Biol. 18: 211-218), sunflower pollen expression genes (Baltz et al. (1992) The Plant Journal 2: 713-721), and rape pollen specific genes (Arnoldo et al. (1992) J. Cell. Biochem, Abstract Y101204).

Stress-inducible promoters include salt/water stress-inducible promoters—such as P5CS (Zang et al. (1997) Plant Sciences 129:81-89), cold-inducible promoters—such as cor15a (Hajela et al. (1990) Plant Physiol .93: 1246-1252), cor15b (Wlihelmetal. (1993) Plant Mol Biol 23: 1073-1077), wsc120 (Ouellet et al. (1998) FEBS Lett. 423-324-328), ci7 (Kirch et al .(1997) PlantMol Biol.33:897-909), ci21A (Schneider et al. (1997) Plant Physiol.113:335-45); desiccation-inducible promoters - eg Trg-31 (Chaudhary et al (1996) Plant Mol.Biol.30:1247-57); osmolyte-inducible promoters—such as Rab17 (Vilardell et al. (1991) Plant Mol.Biol.17:985-93) and osmoproteins (Raghothama et al. (1993) Plant Mol Biol 23:1117-28), and heat-inducible promoters—such as heat shock proteins (Barros et al. (1992) Plant Mol.19:665-75; Marrs et al. (1993) Dev.Genet.14 :27-41) and smHSP (Waters et al. (1996) J. Experimental Botany 47:325-338). Other stress-inducible promoters include rip2 (US Patent 5,332,808 and US Patent Publication 2003/0217393) and rd29a (Yamaguchi-Shinozaki et al. (1993) Mol. Gen. Genetics 236:331-340).

Stress-insensitive promoters may also be used in the methods of the invention. Such promoters and representative examples are further described elsewhere herein.

Nitrogen responsive promoters may also be used in the methods of the invention. Such promoters include, but are not limited to, the 22kDa zein promoter (Spena et al. (1982) EMBO J 1:1589-1594 and Muller et al. (1995) J. Plant Physiol 145:606-613); 19kDa maize Protein promoter (Pedersen et al. (1982) Cell 29:1019-1025); 14kDa zein promoter (Pedersen et al. (1986) J.Biol.Chem.261:6279-6284), b-32 promoter (Lohmer et al. (1991) EMBO J10:617-624) and nitrite reductase (NiR) promoter (Rastogi et al. (1997) Plant Mol Biol.34 (3): 465-76 and Sander et al . (1995) Plant Mol Biol. 27(1): 165-77). For a review of nitrogen-inducible promoter consensus sequences see, eg, Muller et al. (1997) The Plant Journal 12:281-291.

Chemically regulated promoters can also regulate gene expression in plants through the use of exogenous chemical regulators. For this purpose, the promoter may be a chemically inducible promoter, in which gene expression is induced by the use of a chemical substance, or a chemically repressible promoter, in which gene expression is repressed by the use of a chemical substance. Chemically inducible promoters are known in the art, including, but not limited to, the maize In2-2 promoter, which can be activated by benzamide herbicide safeners, and can be used as a hydrophobic electrophile for pre-emergence herbicides. The maize GST promoter activated by the compound, and the tobacco PR-1a promoter activated by salicylic acid. Other chemically regulated promoters of interest include steroid-responsive promoters (see, for example, Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.14 ( 2): glucocorticoid-inducible promoters in 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237 and U.S. Patents 5,814,618 and 5,789,156 ), which is incorporated herein by reference.

Cytokinin-inducible promoters such as the ZmCkx1-2 promoter (US Patent 6,921,815, and pending US Patent Application 11/074,144) may also be used in the methods and compositions of the invention. Such constructs amplify cytokinin synthesis at the developmental stage and/or in the target tissue. Other cytokinin-inducible promoters are described in pending US Patent Application Nos. 11/094,917 and 60/627,394, which are incorporated herein by reference.

Other inducible promoters include heat shock promoters - such as Gmhsp17.5-E (soybean) (Czarnecka et al. (1989) Mol Cell Biol. 9 (8): 3457-3463); Mustard) (Storozhenko et al. (1998) Plant Physiol.118 (3): 1005-1014); Ha hsp 17.7 G4 (Sunflower) (Almoguera et al. (2002) Plant Physiol. 129 (1): 333-341) and maize Hsp70 (Rochester et al. (1986) EMBO J. 5:451-8).

The methods of the invention comprise introducing a polypeptide or polynucleotide into a plant. "Introduction" refers to the manner in which a polynucleotide or polypeptide is present in a plant and enters the interior of a plant cell. The methods of the present invention do not depend on a particular method of introducing sequences into plants, as long as the polynucleotide or polypeptide enters at least one plant cell. Methods for introducing polynucleotides or polypeptides into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

"Stable transformation" means that the target nucleotide construct introduced into the plant is integrated into the plant genome and can be inherited to its progeny. "Transient transformation" means that a sequence is introduced into a plant where it is only temporarily expressed or present.

Transformation procedures and procedures for introducing polypeptide or polynucleotide sequences into plants for effecting transformation may vary depending on the type of plant or plant cell, eg, monocot or dicot. Suitable methods for introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83 :5602-5606), Agrobacterium-mediated transformation (US Patent 5,563,055 and US Patent 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.3: 2717-2722) and ballistic particle acceleration (see for example, US Patent 4,945,050; US Patent 5,879,918; US Patents 5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. . (1988) Biotechnology 6:923-926); Lec1 transformation (WO 00/28058). See also Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onions); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.Biol.27P:175-182 (soybean); Singh et al. (1998) Theor.Appl.Genet.96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Hoque et al. (2005) Plant Cell Tissue & Organ Culture 82 (1): 45-55 (rice); Sreekala et al. (2005) Plant Cell Reports 24 (2): 86-94 (rice); Klein et al. (1988) Proc.Natl.Acad.Sci .USA 85: 4305-4309 (maize); Klein et al. (1988) Biotechnology 6: 559-563 (maize); U.S. Patent 5,240,855; 5,322,783 and 5,324,646; Klein et al. 444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; US Patent 5,736,369 (cereals); Bytebier et al.(1987) Proc.Natl.Acad.Sci.USA 84:5345-5349 (Lily); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed.Chapman et al.(Longman, New York ), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation); D'Halluin et al. (1992) PlantCell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996 ) Nature Biotechnology 14:745-750 (Maize transformation by Agrobacterium tumefaciens); all documents are incorporated herein by reference.

In a specific technical solution, various transient transformation methods for introducing the IPT sequence or IPT promoter sequence of the present invention into plants are provided. Such transient transformation methods include, but are not limited to, directly transferring IPT protein or IPT promoter or variants and fragments thereof into plants, or transferring IPT transcripts into plants. Such methods include, for example, microinjection or particle bombardment. See, e.g., Crossway et al. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107: 775-784, all of which are incorporated herein by reference. Alternatively, the IPT polynucleotide or IPT promoter can be transiently transformed into plants by techniques known in the art. Such techniques include viral vector systems as well as polynucleotide precipitation methods that preclude subsequent release of DNA. Thus, particle-bound DNA can be transcribed, but it is released and integrated into the genome at a greatly reduced frequency. This method involves particle-coated polyethylene (PEI; Sigma #P3143).

In other embodiments, polynucleotides of the invention can be introduced into plants by contacting the plants with viruses or viral nucleic acids. Typically, such methods involve incorporating a nucleotide construct of the invention into a viral DNA or RNA molecule. It will be appreciated that the IPT polynucleotides of the present invention may initially be synthesized as part of a viral polyprotein and then proteolytically processed in vivo or in vitro to produce the desired recombinant protein. In addition, it should be appreciated that promoters used in the present invention may also include promoters transcribed by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing their encoded proteins involving viral DNA or RNA molecules are known in the art. See, eg, U.S. Patents 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931 and Porta et al. (1996) Molecular Biotechnology 5:209-221; incorporated herein by reference.

Methods for inserting polynucleotides into specific sites in the plant genome are known in the art. In one technical solution, the insertion of the polynucleotide at the desired genomic site is accomplished by a site-specific recombination system.参见例如WO99/25821、WO99/25854、WO99/25840、WO99/25855和WO99/25853和美国6,187,994；6,552,248；6,624,297；6,331,661；6,262,341；6,541,231；6,664,108；6,300,545；6,528,700和6,911,575，所有文献作为参考引入本文中. Briefly, a transfer cassette comprising a polynucleotide of the invention is flanked by two non-recombinant recombination sites. The transfer cassette is introduced into plants with stably integrated target sites on the genome flanked by non-recombinogenic recombination sites corresponding to the sites on the transfer cassette. Appropriate recombinases are provided to allow integration of the transfer cassette into the target site. The polynucleotide of interest can therefore integrate into a specific chromosomal location in the plant genome.

Transformed cells can be grown in plants according to conventional methods. See, eg, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can be grown and pollinated with the same transformed plant or with a different plant and progeny expressing the desired phenotypic characteristic identified. Two or more generations can be grown to ensure stable expression and inheritance of the desired phenotypic characteristic, and the seeds harvested to confirm that expression of the desired phenotypic characteristic has been achieved. Thus, the invention provides transformed seeds (also referred to as "transgenic seeds") having a polynucleotide of the invention, eg, an expression cassette of the invention, stably integrated in its genome.

Pedigree breeding begins with a testcross of two genotypes, such as an elite line of interest and another sib line that is complementary to the original line and possesses one or more desired characteristics (i.e., stably integrates a polynucleotide of the invention, have regulatory activity and/or the level of the polypeptide of the present invention, etc.). Other sources may also be included in the inbreeding population if the original parents do not provide all the desired characteristics. In the pedigree approach, superior plants are self-pollinated and screened among successive progeny. In subsequent progeny, the hybrid status is replaced by homologous lines produced by self-pollination and selection. Typically, in a pedigree approach to breeding, 5 or more consecutive progeny are selfed and screened: F1→F2; F2→F3; F3→F4; F4→ _F5 , etc. After a sufficient amount of inbreeding, the subsequent offspring can increase the seeds developed by inbreeding. In a specific embodiment, inbreeding contains homozygous alleles of Yes at about 95% or more of the loci.

In addition to being used to generate backcross transformations, backcrossing can also be used in conjunction with pedigree breeding to alter the target stock line and the heterozygotes produced using the altered stock line. Backcrossing can also be used to transfer one or more specific desired traits from a line - the donor parent - to an inbred recurrent parent - which has essentially all the good agronomic traits but lacks the desired characteristics or features. However, the same procedure can be used to make the progeny have the genotype of the recurrent parent, but at the same time retain many components of the non-recurrent parent by stopping backcrossing early and through selfing and selection. For example, F1 such as commercial heterozygotes are produced. This commercial heterozygote can be backcrossed to one of its parental lines to produce BC1 or BC2. The offspring are selfed and screened so that the newly developed inbred offspring have many characteristics of the recurrent parent as well as several characteristics of the non-recurrent parent. This approach balances the value and strength of new heterozygotes and recurrent parents used in breeding.

Therefore, a technical scheme of the present invention is to prepare the method for the backcross transformation of the target maize inbred line, comprising combining the target maize inbred line with the Any regulated cytokinin levels resulting in a plant phenotype (such plant phenotypes are discussed below) are backcrossed to donor plants for the mutated gene or transgene, and F1 progeny are screened for the mutated gene or transgene conferring the desired trait generation, and backcross the screened F1 progeny plants with the target maize inbred line. The method may further include the step of obtaining the molecular marker profile of the target maize inbred line, and using the molecular marker profile to screen progeny plants having desired characteristics and the molecular marker profile of the target inbred line. In like manner, the method can be achieved by adding a final step of crossing the desired characteristic of the desired characteristic of the maize inbred line of interest to produce F1 heterozygous maize seeds containing the mutant or transgene conferring the desired characteristic. F1 heterozygous seeds are produced.

Recurrent selection is a method of improving plant populations in plant breeding programs. The method is passed on to individual plants by cross-pollination to form progeny. Progeny are grown and screened for dominant progeny by any selection method, including individual plants, half-sib progeny, full-sib progeny, selfed and top-crossed progeny. The screened progeny are cross-pollinated with each other to form another population of progeny. The population is cultivated, and the dominant plants are screened again for mutual cross-pollination. Round selection is a cyclic process, so it can be repeated as many times as necessary. The purpose of recurrent selection is to improve the quality of the population. The improved population is then used as a source of breeding material to obtain inbred lines that can be used as heterozygotes or as parents of synthetic cultivated plants. Synthetic cultivars are the progeny formed by crossing several selected inbred lines.

Hybrid selection is a useful technique when used in combination with molecular marker-enhanced selection. In mixed selection, seeds from individuals are screened according to phenotype and/or genotype. These selected seeds are then mixed together and used to grow the next generation. Mixed selection entails growing plant populations in batches, allowing the plants to self-pollinate, harvesting the seeds in batches, and then using a sample of the batch-harvested seeds to grow the next generation. In addition to self-pollination, direct pollination can also be used as part of a breeding project.

Mutation breeding is a method of introducing new traits into the original germline. Spontaneous or artificially induced mutations can be useful sources of variants for plant breeding. The purpose of artificial mutagenesis is to increase the mutation rate of a desired trait. Mutagenesis rates can be increased by a number of different methods, including temperature, long-term seed storage, tissue culture conditions, radiation—such as X-rays, gamma rays (such as cobalt-60 or cesium-137), neutrons (uranium-235 nuclei in atomic reactors) fission products), beta rays (emanating from radioactive isotopes such as phosphorus-32 or carbon-14), or ultraviolet light (preferably from 2500 to 2900 nm), or chemical mutagens (such as base analogs (5-bromouracil), related compounds (8-Ethoxycaffeine), Antibiotics (Streptonigamicin), Alkylating Agents (Sulfur Mustard, Nitrogen Mustard, Epoxides, Ethylamines, Sulfates, Sulfonates, Sulfolactone), Azide Compounds, hydroxylamine, nitric acid or acridine. When the desired trait is obtained by mutagenesis, the trait can be integrated into the existing germplasm through traditional breeding techniques such as backcrossing. Details of mutation breeding can be found in " Principals of Cultivar Development," Fehr, 1993 Macmillan Publishing Company, the description of which is incorporated herein by reference. Alternatively, mutations made in other lines may be used to generate backcross transformations of the original germline containing the mutation.

The present invention can be used to transform any plant species, including but not limited to monocots and dicots. Examples of target plant species include, but are not limited to, maize (Zea mays), Brassica (e.g., B. napus, B. rapa, B. juncea), especially those species of Brassica used as seed oil feedstock, alfalfa (Medicago sativa ), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vilgare), millet (e.g. Pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), millet (Setariaitalica), dragon claw millet (Eleusine coracana) ), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum ), Yam (Ipomoea batatus), Cassava (Manihot esculenta), Coffee (Coffea spp.), Coconut (Cocos nucifera), Pineapple (Ananas comosus), Citrus (Citrus spp.), Cocoa (Theobroma cacao), Tea (Camellia sinensis), Banana (Musa spp.), Avocado (Persea americana), Fig (Ficus casica), Guava (Psidium guajava), Mango (Mangifera indica), Olive (Oleaeuropaea), Papaya (Carica papaya), Cashews (Anacardium occidentale), macadamia nuts (Macadamia integrifolia), apricots (Prunus amygdalus), beetroot (Beta vulgaris), sugar cane (Saccharum spp.), oak, barley, vegetables, ornamentals and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuces (e.g., Lactucasativa), green beans (Phaseolus vulgaris), green beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the cucumber genus—such as cucumbers (C. sativus ), cantaloupe ( C. cantalupensis) and muskmelon (C. melo). Ornamental plants include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), rose (Rosa spp.), tulip (Tulipa spp.), daffodil (Narcissus spp.), petunia (Petunia hybrida ), carnation (Dianthus caryophyllus), orangutan (Euphorbia pulcherrima) and chrysanthemum.

Conifers that can be used in the practice of the present invention include, for example, pines - such as Pinustaeda, Pinus elliotii, Pinus ponderosa, Pinus contorta and Pinus radiata; (Pseudotsuga menziesii); larch (Tsuga canadensis); spruce (Picea glauca); (Thuja plicata) and Alaskan ponderosa pine (Chamaecyparis nootkatensis). In a specific technical solution, the plant of the present invention is an agricultural crop (such as corn, alfalfa, sunflower brassica, soybean, cotton, safflower, peanut, wheat, millet, tomato, etc.). In other embodiments, corn and soybean plants are preferred, and in still other embodiments, corn plants are preferred.

Other plants of interest include cereals, seed oil plants and leguminous plants that provide the seeds of interest. Target seeds include cereal seeds - eg corn, wheat, barley, rice, sorghum, rye and the like. Seed oil plants include cotton, soybean, safflower, sunflower, canola, corn, alfalfa, palm, coconut, and others. Legumes include beans and peas. Beans include guar, carob, fenugreek, soybeans, lima beans, cowpeas, mung beans, lima beans, broad beans, lentils, chickpeas, and more.

Typically, the invention can be practiced using intermediate host cells to increase the copy number of the cloning vector. As the copy number increases, large numbers of vectors containing the nucleic acid of interest can be isolated for introduction into desired plant cells. In one embodiment, a plant promoter that does not cause expression of the polypeptide in bacteria is used.

The most commonly used prokaryotes are represented by various strains of E. coli, but other microbial strains may also be used. Commonly used prokaryotic regulatory sequences that include transcription initiation within the definition herein, optionally containing an operator, and carrying a ribosome binding sequence include those commonly used promoters such as beta-lactamase (penicillinase) and lactose (lac) promoter system (Chang et al. (1977) Nature 198: 1056), tryptophan (trp) promoter system (Goeddel et al. (1980) Nucleic Acids Res. 8: 4057) and lambda derivatives PL promoter and N-gene ribosome binding site (Shimatake et al. (1981) Nature292: 128). The inclusion of selectable markers has also been used in DNA vectors for transfection of E. coli. Examples of such markers include specific resistance genes to ampicillin, tetracycline or chloramphenicol.

Select a vector that can be introduced into an appropriate host cell. Bacterial vectors are mainly of plasmid or phage origin. Suitable bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If plasmids are used, bacterial cells are transfected with plasmid vector DNA. Expression systems for expressing the proteins of the invention are available from Bacillus and Salmonella (Palva et al. (1983) Gene 22:229-235); Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems, such as yeast, insect cell lines, plant and mammalian cells, are known to those skilled in the art. As described in detail below, polynucleotides of the invention can be expressed in these eukaryotic systems. In some of the techniques discussed below, transformed/transfected plant cells can be used as an expression system to produce the proteins of the invention.

Synthesis of heterologous polynucleotides in yeast is known (Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory). Two widely used eukaryotic protein-producing yeasts are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains and experimental procedures in S. cerevisiae and Pichia pastoris are known in the art and available through commercial suppliers (eg Invitrogen). Suitable vectors generally have expression control sequences, such as a promoter, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences, and other desired elements. Following expression, the proteins of the invention can be isolated from yeast by lysing the cells and using standard protein isolation techniques as listed. Purification can be monitored by Western blot techniques or radioimmunoassays or other standard immunoassay techniques.

The sequences of the invention can also be ligated into various expression vectors for transfection of, for example, cell cultures of mammalian, insect or plant origin. Exemplary cell cultures for the production of polypeptides are mammalian cells. Various suitable host cell lines expressing intact proteins have been developed in the art, including HEK293, BHK21 and CHO cell lines. Expression vectors for these cells include expression regulatory sequences—such as an origin of replication, a promoter (such as the CMV promoter, HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al. (1986) Immunol. Rev.89:49) and essential processing information sites - such as ribosome binding sites, RNA splicing sites, polyadenylation sites (such as SV40 large T Agpoly A sites) and transcription terminator sequences . There are also other animal cells useful for producing proteins of the invention, such as cells from the American Type Culture Collection.

Suitable vectors for expressing the proteins of the invention in insect cells are usually derived from SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines - eg the Schneider cell line (see Schneider (1987) J. Embryol. Exp. Morphol. 27:353-365).

As with yeast, when using higher animal or plant host cells, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences that promote transcriptional splicing are also included. An example of a splice sequence is the VP1 intron from SV40 (Sprague et al. (1983) J. Virol. 45:773-781). In addition, gene sequences that control replication in host cells, such as those found in bovine papillomavirus-type vectors, may also be integrated into the vector (Saveria-Campo (1985) DNA Cloning Vol. II a Practical Approach, D.M. Glover , Ed., IRL Press, Arlington, Virginia, pp. 213-238).

Animal and lower eukaryotic (eg, yeast) host cells can be made available for transfection by a variety of methods. There are several methods for introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of recipient cells with DNA-containing bacterial protoplasts, treatment of recipient cells with DNA-containing liposomes, DEAE dextrin, electroporation, particle gun, and microinjection of DNA directly into cells. Transfected cells are cultured by methods known in the art (Kuchler (1997) Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc.).

II. Regulation of Prenyltransferase Polypeptide Concentration and/or Activity

Methods of modulating the concentration and/or activity of a polypeptide of the invention in a plant are provided. Typically, the IPT polypeptide concentration and/or activity is increased or decreased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, relative to a native control plant, plant part or cell without the introduced sequence %, 70%, 80% or 90% or more. Modulation of concentration and/or activity can occur at one or more stages of development. In a specific technical solution, the polypeptide of the present invention is regulated in monocotyledonous plants such as maize.

The expression level of an IPT polypeptide can be determined directly, eg, by measuring the level of the IPT polypeptide in a plant, or indirectly, eg, by measuring cytokinin synthesis activity in a plant. Methods for assaying cytokinin synthesis activity are described elsewhere herein.

In a specific technical solution, the polypeptide or polynucleotide of the present invention is introduced into plant cells. Plant cells are then screened for the introduced sequences of the invention by methods known to those skilled in the art, such as, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis or phenotypic analysis. Plants or plant parts changed or modified by the aforementioned technical solutions are grown for a certain period of time under plant-forming conditions, so as to fully regulate the concentration and/or activity of the polypeptide of the present invention in plants. Planting conditions are known in the art and discussed briefly elsewhere herein.

It is also recognized that polypeptide levels and/or activity can be modulated by the use of polynucleotides that do not direct protein or RNA expression in transformed plants. For example, the polynucleotides of the invention can be used to design polynucleotide constructs - which can be used in methods of altering or mutating the nucleotide sequence of the genome of an organism. Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNA:DNA mutation vectors, RNA:DNA repair vectors, mixed duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinant oligonucleotides. Nucleotides. Such nucleotide constructs and methods of use are known in the art. See US Patents 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984; all incorporated herein by reference. See also WO 98/49350, WO 99/07865, WO 99/25821 and Beetham et al. (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; incorporated herein by reference.

The method of the invention therefore does not depend on whether the complete polynucleotide is integrated in the genome, as long as the plant or its cells are altered by the polynucleotide introduced into the cell. In one technical solution of the present invention, the genome can be altered by introducing polynucleotides into cells. For example, a polynucleotide or any portion thereof can be integrated into the plant genome. Changes in the genome include, but are not limited to, additions, deletions, and substitutions of nucleotides in the genome. While the method of the present invention does not rely on any specific number of nucleotide additions, deletions and substitutions, it is recognized that such additions, deletions or substitutions should contain at least one nucleotide.

It is further shown that the level and/or activity of IPT sequences can be modulated by introducing sequences that act only at specific developmental stages, and turning off their effects at other stages when their expression is not required. IPT expression can be controlled through the use of inducible or tissue-preferred promoters. Alternatively, the gene can be inverted or deleted by site-specific recombinases, transposons, or recombination systems, which can turn expression of the IPT sequence on or off.

"Tested plant or plant cell" means a plant or plant cell in which a genetic alteration, such as transformation, has the same effect as the gene of interest, or is derived from, and contains such an alteration. A "control" or "control plant" or "control plant cell" provides a point of reference for determining a change in phenotype of a test plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material that was subjected to the genetic changes resulting in the test plant or cell; (b) of the same genotype as the starting material. genotyped plants or plant cells, but transformed with a null construct (i.e., a construct that has no known effect on the trait of interest—such as a construct containing a marker gene); (c) in the test plant or plant cell progeny (d) a plant or plant cell genetically identical to the test plant or plant cell, but not exposed to conditions or stimuli that induce expression of the gene of interest, or (e) in the absence of expression of the target gene The test plant or the plant cell itself under the condition of the gene.

In this case, for example, alterations in cytokinin levels, including changes in absolute cytokinin amounts, cytokinin ratios, cytokinin activity, or cytokinin distribution, or changes in plant or plant cell phenotype—such as Flowering time, seed fixation, branching, senescence, stress tolerance or root size may be determined by comparing test plants or plant cells to control plants or plant cells.

A Increase the activity and/or concentration of prenyltransferase polypeptide

Methods of increasing the activity and/or increasing the concentration of an IPT polypeptide of the invention are provided. An increase in the concentration and/or activity of an IPT polypeptide of the invention can be achieved by providing an IPT polypeptide to a plant. As discussed elsewhere herein, a number of methods of providing polypeptides to plants are known in the art, including, but not limited to, direct introduction into plants of polypeptides, and introduction into plants (transient or stable) of genes encoding Polynucleotide constructs of peptides with synthetic activity. It is also known that the methods of the invention can use polynucleotides that are not capable of directing protein or RNA expression in transformed plants. Accordingly, IPT polypeptide levels and/or activity can be increased by altering the gene encoding the IPT polypeptide or its promoter. See, eg, Kmiec, US Patent 5,565,350; Zarling et al., PCT/US93/03868. Accordingly, mutant plants carrying a mutation in the ITP gene are provided, wherein the mutation increases expression of the IPT gene or increases the cytokinin synthesis activity of the encoded IPT polypeptide. As described elsewhere herein, methods for measuring increased protein concentration or increased cytokinin synthesis activity are known.

B Reduce prenyltransferase polypeptide activity and/or concentration

Methods are provided for reducing or eliminating the activity and/or concentration of an IPT polypeptide by using an expression cassette that expresses a polynucleotide that inhibits expression of the IPT polypeptide. A polynucleotide can inhibit IPT polypeptide expression directly by inhibiting translation of IPT polypeptide messenger RNA, or indirectly by encoding a molecule that inhibits transcription or translation of an IPT polypeptide gene encoding an IPT polypeptide. Methods for inhibiting or abrogating gene expression in plants are known in the art, and any method may be used in the present invention to inhibit expression of an IPT polypeptide.

According to the present invention, expression of an IPT polypeptide is inhibited if the level of the IPT polypeptide is statistically lower than the level of the same IPT polypeptide in a plant that has not been genetically altered or mutated to inhibit expression of the IPT polypeptide. In a particular technical solution of the invention, according to the invention, the protein level of the IPT polypeptide in the altered plant is 95%, 90% lower than the protein level of the same IPT polypeptide in the plant which has not been mutated or genetically altered to inhibit the expression of the IPT polypeptide. %, 80% lower, 70% lower, 60% lower, 50% lower, 40% lower, 30% lower, 20% lower, 10% lower, or 5% lower. The expression level of an IPT polypeptide can be determined directly, eg, by measuring the level of IPT polypeptide expressed in a cell or plant, or indirectly, eg, by measuring cytokinin synthesis activity in a cell or plant. Methods for assaying the cytokinin synthesis activity of IPT polypeptides are described elsewhere herein.

In other technical solutions of the present invention, the activity of one or more IPT polypeptides is reduced or eliminated by transforming plant cells with an expression cassette containing a polynucleotide encoding a polypeptide that inhibits the activity of one or more IPT polypeptides. According to the present invention, if the IPT cytokinin synthesis activity is statistically lower than the cytokinin synthesis activity of the same IPT polypeptide in plants that have not been genetically altered to inhibit the IPT polypeptide cytokinin synthesis activity, the cytokinin synthesis activity of the IPT polypeptide is activity is inhibited. In a particular embodiment of the invention, according to the invention, the cytokinin synthesis activity of the IPT polypeptide is 95% lower in the altered plant than the cytokinin synthesis activity of the same IPT polypeptide in a plant that has not been genetically altered to inhibit the expression of the IPT polypeptide %, 90% lower, 80% lower, 70% lower, 60% lower, 50% lower, 40% lower, 30% lower, 20% lower, 10% lower or less than 5% lower. According to the present invention, the cytokinin synthesis activity of an IPT polypeptide is "depleted" when detected in the absence of detection methods as described elsewhere herein. Methods for assaying the cytokinin synthesis activity of IPT polypeptides are described elsewhere herein.

In other technical solutions, the activity of the IPT polypeptide can be reduced or eliminated by destroying the gene encoding the IPT polypeptide. The present invention includes mutant plants carrying a mutation in the IPT gene, wherein the mutation reduces the expression of the IPT gene or inhibits the cytokinin synthesis activity of the encoded IPT polypeptide.

Accordingly, various methods can be used to reduce or eliminate the activity of IPT polypeptides. More than one method can be used to reduce the activity of a single IPT polypeptide. Additionally, various approaches can be used in combination to reduce or eliminate the activity of two or more different IPT polypeptides.

Non-limiting examples of methods for reducing or eliminating expression of an IPT polypeptide are given below.

1. Polynucleotide-based methods

In some technical solutions of the present invention, plant cells are transformed with an expression cassette containing a polynucleotide capable of expressing an expression-inhibiting IPT sequence. The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, an expression cassette that expresses a polynucleotide that inhibits the expression of at least one IPT sequence is an expression cassette that produces an RNA molecule that inhibits the transcription and/or translation of at least one IPT polypeptide, for purposes of the present invention. "Expressing" or "producing" a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while "expressing" or "producing" a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce protein or polypeptide.

Examples of polynucleotides that inhibit expression of IPT sequences are given below.

i. Sense suppression/co-suppression

In some technical solutions of the present invention, the expression of IPT polypeptides can be suppressed by sense suppression or co-suppression. For co-suppression, the expression cassette is designed to express in the "sense" orientation an RNA molecule corresponding to all or part of the messenger RNA encoding the IPT polypeptide. Overexpression of RNA molecules can result in decreased expression of native genes. Multiple plant lines transformed with the co-suppression expression cassette are thus screened to identify those that exhibit the greatest degree of inhibition of IPT polypeptide expression.

The polynucleotide used for co-suppression may correspond to all or part of a sequence encoding an IPT polypeptide, all or part of the 5' and/or 3' untranslated region of an IPT polypeptide transcript, or the coding sequence of all or part of a transcript encoding an IPT polypeptide. sequences and untranslated regions. In certain embodiments, wherein the polynucleotide comprises all or part of the IPT polypeptide coding region, the expression cassette is designed to delete the start codon of the polynucleotide so that no protein product is transcribed.

Cosuppression can be used to suppress plant gene expression to produce plants in which the levels of proteins encoded by these genes are undetectable. See, eg, Broin et al. (2002) Plant Cell 14:1417-1432. Cosuppression can also be used to suppress the expression of multiple proteins in the same plant. See, eg, US Patent 5,942,657. In Flavell et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3490-3496; Jorgensen et al. (1996) Plant Mol. Biol. 31: 957-973; Johansen and Carrington (2001) Plant Physiol. 126: 930-938; Broin et al. (2002) Plant Cell 14: 1417-1432; Stoutjesdijket al (2002) Plant Physiol 129: 1723-1731; Yu et al. (2003) Phytochemistry 63: 753-763 and US Patent Methods for inhibiting expression of endogenous genes in plants using cosuppression are described in 5,034,323, 5,283,184 and 5,942,657, all incorporated herein by reference. The efficiency of cosuppression can be increased by including a poly-dT region 3' to the sense sequence of the expression cassette and 5' to the polyadenylation signal. See US Patent Publication 20020048814, incorporated herein by reference. Typically, such nucleotide sequences have substantial sequence identity to the sequence of the endogenous gene transcript, preferably greater than 65% sequence identity, more preferably greater than 85% sequence identity, most preferably greater than 95% sequence identity. See US Patents 5,283,184 and 5,034,323; incorporated herein by reference.

ii. Antisense suppression

In some technical solutions of the present invention, the inhibition of IPT polypeptide expression can be performed by antisense inhibition. For antisense suppression, the expression cassette is designed to express an RNA molecule that is complementary to all or part of the messenger RNA encoding the IPT polypeptide. Overexpression of antisense RNA molecules can reduce the expression of native genes. Multiple plant lines transformed with the antisense suppression expression cassette were thereby screened and those exhibiting the greatest suppression of IPT polypeptide expression identified.

A polynucleotide for antisense suppression may correspond to all or part of the complement of a sequence encoding an IPT polypeptide, the complement of all or part of the 5' and/or 3' untranslated regions of an IPT polypeptide transcript, or all or part of a sequence encoding an IPT polypeptide. The coding sequence and the complement of the untranslated region of the IPT polypeptide transcript. In addition, antisense polynucleotides can be fully complementary (ie, 100% identical to the complement of the target sequence) or partially complementary (ie, less than 100% identical to the complement of the target sequence) to the target sequence. Antisense suppression can be used to suppress the expression of multiple proteins in the same plant. See, eg, US Patent 5,942,657. Alternatively, antisense nucleotide moieties can be used to disrupt the expression of a gene of interest. Typically sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or more nucleotides are used. Methods for inhibiting expression of endogenous genes in plants using antisense suppression are described, for example, in Liu et al (2002) Plant Physiol. 129:1732-1743 and U.S. Patent Nos. 5,759,829 and 5,942,657, each of which is incorporated herein by reference. The efficiency of antisense suppression can be increased by including a poly-dT region 3' to the antisense sequence of the expression cassette and 5' to the polyadenylation signal. See US Patent Publication 20020048814, incorporated herein by reference.

iii. Stranded RNA interference

In some technical solutions of the present invention, the inhibition of IPT polypeptide expression can be achieved by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule similar to the aforementioned co-suppression and an antisense RNA molecule fully or partially complementary to the sense RNA molecule are expressed in the same cell, thereby inhibiting the expression of the corresponding endogenous messenger RNA.

Expression of sense and antisense molecules can be achieved by designing expression cassettes containing sense and antisense sequences. Alternatively, different expression cassettes can be used for sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or cassettes are then screened and the plant line exhibiting the greatest inhibition of IPT polypeptide expression identified. In Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-13964; Liu et al. (2002) Plant Physiol. 129: 1732-1743 and WO 99/49029, WO 99/53050, WO 99 Methods for inhibiting expression of endogenous plant genes using dsRNA interference are described in WO 00/49031 and WO 00/49035; each of which is incorporated herein by reference.

iv. Hairpin RNA interference and intron-containing hairpin RNA interference

In some technical solutions of the present invention, the expression inhibition of one or more IPT polypeptides can be achieved by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly effective at inhibiting endogenous gene expression. See Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38, incorporated herein.

. For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure containing a single-stranded loop region and a base-paired stem. The base-paired stem region contains a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-pairing stem region of the molecule often determines the specificity of RNA interference. hpRNA molecules can efficiently inhibit the expression of endogenous genes, and the RNA interference induced by them can be inherited to plant progeny. See, eg, Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129: 1723-1731 and Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4:29-38. In eg Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129: 1723-1731; : 29-38; Pandolfini et al. BMC Biotechnology 3: 7 and US Patent Publication 20030175965 describe methods for inhibiting or silencing gene expression using hpRNA interference; each of which is incorporated herein by reference. A method for transient detection of hpRNA construct efficiency is described in Panstruga et al. (2003) Mol. Biol. Rep. 30: 135-140, incorporated herein by reference.

Alternatively, the base paired stem region may correspond to a region of a promoter sequence that controls expression of the gene to be suppressed. Transcriptional gene silencing (TGS) can be achieved by hpRNA constructs in which the inverted repeat of the hairpin has sequence identity to the promoter driving expression of the gene being silenced. Processing of hpRNAs into short RNAs that can interact with cognate promoter regions can trigger degradation or methylation, resulting in silencing (Aufsatz et al. (2002) PNAS 99(Suppl.4): 16499-16506; Mette et al. al. (2000) EMBO J 19(19):5194-5201).

For ihpRNAs, interfering molecules have the same general structure as hpRNAs, but the RNA molecules additionally contain introns that can be spliced in cells expressing ihpRNAs. The use of introns reduces the size of the post-shear loop in hairpin RNA molecules, thereby increasing interference efficiency. See, eg, Smith et al. (2000) Nature 407:319-320. In fact, Smith et al. achieved 100% suppression of endogenous gene expression using ihpRNA-mediated interference. In e.g. Smith et al. (2000) Nature 407: 319-320; Wesley et al. (2001) Plant J. 27: 581-590; Wang and Waterhouse (2001) Curr. Opin. Plant Biol. 5: 146-150; Waterhouse and Helliwell (2003) Nat.Rev.Genet.4: 29-38; Helliwell and Waterhouse (2003) Methods 30: 289-295 and US Patent Publication 20030180945 describe methods for inhibiting endogenous plant gene expression by ihpRNA interference, Each document is incorporated herein by reference.

The expression cassette for hpRNA interference can also be designed so that the sense and antisense sequences do not correspond to endogenous RNA. In this technical solution, the sense and antisense sequences are located on both sides of a loop sequence containing the nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. The specificity of RNA interference is thus determined by the loop region. See for example WO 02/00904, incorporated herein by reference.

v. Amplicon-mediated interference

Amplicon expression cassettes contain plant virus-derived sequences that contain all or part of the gene of interest, but usually none of the genes of the native virus. Viral sequences in the transcript of the expression cassette allow the transcript to direct its own replication. Transcripts produced from the amplicon may be sense or antisense relative to the target sequence (ie, the messenger RNA of the IPT polypeptide). Methods for suppressing expression of endogenous plant genes using amplicons are described, for example, in Angell and Baulcombe (1997) EMBO J.16:3675-3684; Angelland Baulcombe (1999) Plant J.20:357-362 and U.S. Patent 6,646,805, Each document is incorporated herein by reference.

vi. Ribozyme

In some technical solutions, the polynucleotide expressed by the expression cassette of the present invention is catalytic RNA or has ribozyme activity against the messenger RNA of the IPT polypeptide. Thus, polynucleotides can degrade endogenous messenger RNA, resulting in reduced expression of the IPT polypeptide. Such methods are described, for example, in US Patent 4,987,071, incorporated herein by reference.

vii. Small interfering RNA or microRNA

In some technical solutions of the present invention, the inhibition of the expression of one or more IPT polypeptides can be achieved by RNA interference expressing genes encoding microRNAs (miRNAs). miRNAs are regulatory factors containing about 22 ribonucleotides. miRNAs can efficiently inhibit the expression of endogenous genes. See, eg, Javier et al. (2003) Nature 425:257-263, incorporated herein by reference.

For miRNA interference, expression cassettes are designed to express RNA molecules that mimic endogenous miRNA genes. The miRNA gene encodes an RNA that forms a hairpin structure of 22 nucleotides complementary to another endogenous gene (target sequence). For inhibiting IPT polypeptide expression, the 22-nucleotide sequence is selected from an IPT polypeptide transcript sequence, and contains 22 nucleotides encoding said IPT polypeptide sequence in a sense orientation, and a corresponding antisense sequence complementary to the sense sequence of 21 nucleotides. miRNA molecules can efficiently inhibit the expression of endogenous genes, and the RNA interference induced by them can be inherited to plant progeny.

2. Peptide-based gene expression inhibition

In one technical solution, the polynucleotide encodes a zinc finger protein that can bind to a gene encoding an IPT polypeptide, thereby reducing gene expression. In a specific technical solution, the zinc finger protein is combined with the regulatory region of the IPT polypeptide gene. In other technical schemes, the zinc finger protein binds to the messenger RNA encoding the IPT polypeptide and inhibits its translation. Methods for selecting zinc finger protein target sites are described, eg, in US Patent 6,453,242, and methods of inhibiting plant gene expression using zinc finger proteins are described, eg, in US Patent Publication 20030037355; each incorporated herein by reference.

3. Peptide-based protein activity inhibition

In some technical solutions of the present invention, the polynucleotide encodes an antibody that binds to at least one IPT polypeptide and reduces the cytokinin synthesis activity of the IPT polypeptide. In another technical solution, antibody binding can lead to increased turnover of antibody-IPT polypeptide complexes through cellular quality control mechanisms. Expression of antibodies in plant cells and molecular pathways for inhibition by expression in plant cells and binding of antibodies to proteins are known in the art. See, eg, Conrad and Sonnewald (2003) Nature Biotech. 21:35-36, incorporated herein by reference.

4. Gene disruption

In some technical solutions of the present invention, the activity of the IPT polypeptide is reduced or eliminated by destroying the gene encoding the IPT polypeptide. The gene encoding the IPT polypeptide can be disrupted by any method known in the art. For example, in one solution, genes are disrupted by transposon tagging. In another technical solution, the gene is disrupted by mutagenizing the plants by random or directed mutagenesis, and selecting plants for reduced IPT activity.

i. Transposon Marking

In one technical solution of the present invention, the cytokinin synthesis activity of one or more IPT polypeptides is reduced or eliminated using transposon tags. Transposon tagging involves the insertion of a transposon into an endogenous IPT gene, thereby reducing or eliminating IPT polypeptide expression. "IPT gene" refers to a gene encoding an IPT polypeptide of the present invention.

In this technical solution, the expression of one or more IPT polypeptides is reduced or eliminated by inserting transposons into the gene regulatory region or coding region encoding IPT polypeptides. Transposons located in exons, introns, 5' or 3' untranslated sequences, promoters, or any other regulatory sequences of the IPT polypeptide gene can be used to reduce or eliminate the expression and/or activity of the encoded IPT polypeptide.

Methods for transposon tagging of specific genes in plants are known in the art. See, eg, Maes et al. (1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett. 179:53-59; Meissner et al. (2000) Plant J. 22:265-274 ; Phogat et al. (2000) J.Biosci.25: 57-63; Walbot (2000) Curr. Opin. Plant Biol. 2: 103-107; Gai et al. (2000) Nucleic Acids Res. 28: 94- 96; Fitzmaurice et al. (1999) Genetics 153:1919-1928). In addition, the TUSC process for screening for Mu insertions in selected genes is described in Bensen et al. (1995) Plant Cell 7: 75-84; Mena et al. (1996) Science 274: 1537-1540 and US Patent 5,962,764, each The documents are all incorporated herein by reference.

ii. Mutant plants with reduced activity

Other methods of reducing or eliminating expression of endogenous genes in plants are also known in the art and can be applied in a similar manner in the present invention. These methods include other forms of mutagenesis - such as methanesulfonic acid-induced mutagenesis used in reverse genetics manipulations (by PCR) to identify plant lines from which endogenous genes have been removed, deletion mutagenesis and fast neutron deletion mutagenesis . See Ohshima et al. (1998) Virology 243: 472-481 for examples of these methods; Okubara et al. (1994) Genetics 137: 867-874 and Quesada et al. (2000) Genetics 154: 421-436; All are incorporated herein by reference. In addition, the method TILLING (Target-Induced Localized Lesions In Genome) for rapid automated screening of chemically induced mutations using denaturing HPLC or selective endonuclease digestion of selected PCR products can also be applied in the present invention. See McCallum et al. (2000) Nat. Biotechnol. 18:455-457, incorporated herein by reference.

Mutations that affect gene expression or interfere with the function of the encoded protein (IPT activity) are known in the art. Insertion mutations in exons of genes often result in nonsense mutations. Mutations at conserved residues are very effective at inhibiting the cytokinin synthesis activity of the encoded protein. Conserved residues in plant IPT polypeptides suitable for mutation for the purpose of abolishing IPT activity have been described. See eg Figure 1. Such mutants can be isolated according to known procedures, and mutations at different IPT sites can be stacked by genetic crosses. See, eg, Gruis et al. (2002) Plant Cell 14:2863-2882.

In another technical solution of the present invention, dominant mutations can be used to initiate RNA silencing due to gene inversion and recombination of repeated gene loci. See, eg, Kusaba et al. (2003) Plant Cell 15: 1455-1467.

The invention encompasses other methods of reducing or eliminating the activity of one or more IPT polypeptides. Other methods of altering or mutating genomic nucleotide sequences in plants are known in the art, including, but not limited to, the use of RNA:DNA vectors, RNA:DNA mutation vectors, RNA:DNA repair vectors, mixed double-stranded oligonucleotides oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinant oligonucleotides. Such vectors and methods of use are known in the art. See, eg, US Patents 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984; each incorporated herein by reference. See also WO 98/49350, WO 99/07865, WO 99/25821 and Beetham et al. (1999) Proc. Natl. Acad. Scl. USA 96:8774-8778; each incorporated herein by reference.

III. Modulation of Cytokinin Levels and/or Activity

"Cytokinins" as used herein refers to a class or members of a plant-specific hormone that plays a central role in the cell cycle and affects various developmental programs. Cytokinins contain N ⁶ -substituted purine derivatives. Representative cytokinins include isopentenyladenosine (N ⁶ -(Δ ² -isopentenyl)adenosine (hereinafter referred to as iP), zeatin (6-(4-hydroxy-3-methylbutyl-trans Formulas - 2-alkenylamino)purine) (hereinafter Z) and dihydrozeatin (DZ). Free bases and their ribose sugars (iPR, ZR and DZR) are active compounds. Other cytokinins are known. See, eg, US Patent 5,211,738 and Keiber et al. (2002) Cytokinins, The Arabidopsis Book, American Society of Plant Biologists, both incorporated herein by reference.

"Modulating cytokinin levels"includes a significant decrease or increase in the level and/or activity of any cytokinin in a plant when compared to a control plant. For example, modulating the level and/or activity comprises increasing or decreasing the total cytokinin content by about 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20% compared to a control plant or plant part , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. Alternatively, the modulating level and/or activity of cytokinins in a plant or plant part may comprise an increase or decrease in the level and/or activity of a cytokinin by about 0.2 fold, 0.5 fold, 2 fold, compared to a control plant or plant part. 4x, 8x, 16x, 32x or more.

It is also recognized that modulation of cytokinin levels/activity does not require an increase/decrease in total cytokinin levels and/or activity, but includes changes in the distribution of cytokinins in tissues. In addition, modulation of cytokinin levels/activity does not require total cytokinin increases/decreases, but involves changes in the ratio of various cytokinin derivatives. For example, the ratio of various cytokinin derivatives, such as isopentenyladenosine-type, zeatin-type or dihydrozeatin-type cytokinins, etc. is altered compared to control plants, thereby regulating the plant or plant part. Cytokinin levels/activity.

Methods for determining regulation of cytokinin levels and/or activity are known in the art. For example, representative methods for cytokinin extraction, immunopurification, HPLC separation, and quantification by ELISA methods are described, for example, in Faiss et al. (1997) Plant J. 12:401-415. See also Werner et al. (2001) PNAS 98: 10487-10492) and Dewitte et al. (1999) Plant Physiol. 119: 111-121. Each document is incorporated herein by reference. As discussed elsewhere herein, modulation of cytokinin levels and/or activity can further be detected by monitoring specific plant phenotypes. This phenotype is described elsewhere herein.

In certain methods, the level and/or activity of a cytokinin is increased in a plant by increasing the level or activity of an IPT polypeptide in the plant. Methods of increasing the level and/or activity of an IPT polypeptide in a plant are described elsewhere herein. Briefly, these methods involve introducing into a plant an IPT polypeptide of the invention, thereby increasing the level and/or activity of the IPT polypeptide. In other technical solutions, by introducing into the plant a polynucleotide containing the IPT nucleotide sequence of the present invention, expressing the IPT sequence, thereby increasing the level of cytokinin and/or in the plant or plant part when compared with the control plant or active mode, an IPT nucleotide sequence encoding an IPT polypeptide is provided. In certain embodiments, the IPT nucleotide construct introduced into the plant is stably integrated in the plant genome.

In other methods, the level and/or activity of a cytokinin is reduced in a plant by reducing the level and/or activity of one or more IPT polypeptides in the plant. This approach is described elsewhere herein. In this method, an IPT nucleotide sequence is introduced into a plant and expression of the IPT nucleotide sequence reduces the activity of the IPT polypeptide such that cytokinin is present in the plant or plant part when compared to a control plant or plant part. Levels and/or activity are reduced. In other embodiments, the IPT construct introduced into the plant is stably integrated in the plant genome.

As noted above, the skilled artisan is aware of the use of suitable promoters to modulate cytokinin levels/activity in plants, exemplary promoters for this approach are described elsewhere herein.

Accordingly, the present invention further provides plants having modulated cytokinin levels/activity when compared to cytokinin levels/activity in control plants. In one embodiment, the plant of the invention has an increased level/activity of an IPT polypeptide of the invention, thereby having an increased level/activity of a cytokinin. In other embodiments, plants of the invention have reduced or removed levels of IPT polypeptides of the invention, thereby having reduced levels/activity of cytokinins. In a particular technical solution, such plants have stably integrated into their genome a nucleic acid molecule comprising the IPT nucleotide sequence of the invention linked to a promoter that initiates expression in the plant cell.

IV. Regulation of Root Development

Methods of modulating root development in plants are provided. By "modulating root development" is meant any change in the process of root development of a plant when compared to a control plant. Such changes in root development include, but are not limited to, changes in primary root growth rate, root fresh weight, extent of lateral and adventitious root formation, vasculature, meristem development, or radial expansion.

Methods of modulating root development in plants are provided. Methods include modulating the level and/or activity of an IPT polypeptide in a plant. In one method, an IPT sequence of the invention is introduced into a plant. In another method, by introducing a polynucleotide containing the IPT nucleotide sequence of the present invention (which may be a fragment of the full-length IPT sequence nucleotide) into the plant, expressing the IPT sequence, thereby regulating root development to provide the IPT nucleotide sequence. In other methods, an IPT nucleotide construct introduced into a plant is stably integrated into the plant genome.

In other methods, root development is modulated by reducing the level or activity of an IPT polypeptide in a plant. This method includes introducing IPT nucleotide sequences into plants, and reducing the activity of IPT polypeptides. In certain methods, the IPT nucleotide construct introduced into a plant is stably integrated into the plant genome. A decrease in cytokinin synthesis activity may result in at least one or more of the following changes in root development, including, but not limited to, having larger root meristems, increased root growth, enhanced radial Dilation, enhanced vasculature, increased root branching, more adventitious roots and/or increased root fresh weight.

"Root growth" as used herein includes all aspects of growth in both monocotyledonous and dicotyledonous plants of the different parts that make up the root system at different stages of root development. It is understood that enhanced root growth may be one or more parts thereof, including enhanced growth of primary roots, lateral roots, adventitious roots, and the like. Methods for determining such developmental changes in the root system are known in the art. See, eg, US Patent Application 2003/0074698 and Werner et al. (2001) PNAS 18:10487-10492, both of which are incorporated herein by reference.

As mentioned above, the skilled person will know to use suitable promoters to regulate root development in plants. Exemplary promoters of this approach include constitutive promoters and root-biased promoters. Exemplary root-biased promoters are described elsewhere herein.

Stimulating root growth and increasing root weight by reducing IPT polypeptide activity and/or levels can be used to improve lodging tolerance of plants. The term "lodging resistance" or "lodging tolerance" refers to the ability of a plant to anchor itself in the soil. For plants with an erect or semi-erect growth habit, the term also refers to the ability to maintain an upright position under adverse environmental conditions. This property concerns the volume, depth and morphology of the root system. In addition, stimulating root growth and increasing root weight by reducing IPT polypeptide levels and/or activity at appropriate developmental stages can be used to promote propagation of explants in vitro.

Increased root biomass and/or altered root architecture can be used to improve the nitrogen use efficiency of plants. Such improved efficiency can result, for example, in increased plant biomass and/or seed yield when available nitrogen is at present levels, or in maintaining plant biomass and/or seed yield when available nitrogen is limited. There are thus agronomic and/or environmental advantages.

Additionally, higher root biomass due to reduced IPT polypeptide levels and/or activity has an indirect effect on the production of compounds produced by root cells or transgenic root cells or cell cultures of said transgenic root cells. An example of a compound of interest produced in root cultures is shikonin, the production of which can be conveniently increased by the method described.

Accordingly, the present invention further provides plants having modulated root development compared to root development of control plants. In certain embodiments, the plants of the invention have reduced levels and/or activity of the IPT polypeptides of the invention, and enhanced root growth and/or root biomass. In a specific technical solution, a nucleic acid molecule comprising the IPT nucleotide sequence of the present invention operably linked to a promoter that promotes expression in a plant cell is stably integrated in the genome of such a plant.

V. Regulation of shoot and leaf development

Methods of modulating vegetative tissue growth in plants are provided. In one embodiment, shoot and leaf development in the plant is regulated. "Modulating shoot and/or leaf development" refers to any change in plant shoot and/or leaf development when compared to a control plant or plant part. Such changes in shoot and/or leaf development include, but are not limited to, changes in root meristem development, leaf number, leaf condition, leaf and stem vasculature, internode length, and leaf senescence. As used herein, "leaf development" and "shoot development" include all aspects of the growth of the different parts making up the leaf system and the shoot system, respectively, at different stages of their development in monocotyledonous and dicotyledonous plants. Methods for measuring developmental changes in such shoot and leaf systems are known in the art. See, eg, Werner et al. (2001) PNAS 98:10487-10492 and US Patent Application 2003/0074698, each of which is incorporated herein by reference.

Methods of modulating shoot and/or leaf development in plants include modulating the activity and/or level of an IPT polypeptide of the invention. In one technical solution, the IPT sequence of the present invention is provided. In other technical solutions, the IPT nucleotide sequence is provided by introducing the polynucleotide containing the IPT nucleotide sequence of the present invention into the plant, expressing the IPT sequence, and regulating the development of shoots and/or leaves. In other methods, an IPT nucleotide construct introduced into a plant is stably integrated into the plant genome.

In a specific technical solution, the development of shoots or leaves is regulated by reducing the level and/or activity of IPT polypeptides in plants. A decrease in IPT activity can result in one or more changes in shoot and/or leaf development, including, but not limited to, smaller apical meristems, reduced leaf number, reduced leaf surface area, reduced veins compared to control plants tube organization, shorter internodes and stunted growth, and accelerated leaf senescence.

As mentioned above, the skilled person will know that shoot and leaf development in plants can be regulated using appropriate promoters. Exemplary promoters of this technique include constitutive promoters, shoot-biased promoters, shoot-meristem-biased promoters, senescence-activated promoters, stress-inducible promoters, root-biased promoters, Nitrogen-inducible and leaf-biased promoters. Example promoters are described elsewhere herein.

Reducing cytokinin synthesis activity in plants generally results in shorter internodes and dwarf growth. Therefore, the method of the present invention can be used to generate dwarf plants. In addition, as described above, modulation of cytokinin synthesis activity in plants can regulate root and shoot growth. Accordingly, the present invention further provides a method of altering the ratio of roots to shoots.

Shoot or leaf development can further be modulated by increasing the level and/or activity of the IPT polypeptide in the plant. Increased IPT activity can result in one or more changes in shoot and/or leaf development, including, but not limited to, increased leaf number, increased leaf surface area, increased vascular organization, increased shoot formation, longer nodes compared to control plants time, improved growth, improved plant yield and vigor and delayed leaf senescence.

In one solution, the plant's tolerance to waterlogging is increased. Waterlogging is a serious environmental stress that affects plant growth and yield. Waterlogging can lead to premature senescence, causing leaf wilting, necrosis, defoliation, cessation of growth and reduced yields. Cytokinin can regulate aging by increasing the level/activity of IPT polypeptide in plants, and the invention improves the tolerance of plants to various environmental stresses including waterlogging. Delayed senescence is conducive to expanding the adaptability of crops to maturity, improving the shelf life of potted plants, and extending the vase life of cut flowers.

In other embodiments, methods for regulating shoot regeneration in callus are provided. In this method, increasing the level and/or activity of the IPT polypeptide will increase the level of cytokinins in the plant. Thus, reduced levels of exogenous growth regulators (ie, cytokinins) or absence of exogenous cytokinins in the medium enhanced shoot regeneration in callus. Thus, in one aspect of the invention, increased levels and/or activity of IPT sequences can be used to overcome poor shoot growth tendencies in certain species in which the success and speed of transgenic technology is limited. In addition, a variety of branch induction can be carried out on commercial crops to produce as many branches as possible. Thus, methods for increasing the rate of regeneration of transformations are provided. In a particular technical solution, the IPT sequence is under the control of an inducible promoter (eg heat shock promoter, chemical inducible promoter). Other inducible promoters are known in the art and discussed elsewhere herein.

Methods for establishing callus tissue from explants are known. For example, roots, stems, shoots and sterile shoot shoots are a few tissue sources that can be used to induce callus formation. Young, growing tissues (eg, young leaves, roots, meristems, or other tissues) are typically used, but not required. Callus formation is controlled by growth-regulating substances (auuxins and cytokinins) in the medium. The specific concentrations of plant regulators required to induce callus formation vary between species and even depend on the source of the explants. In some cases, different growth substances (eg 2,4-D or NAA) or combinations thereof may be used in the assay since certain species do not respond to particular growth regulators. In addition, culture conditions (ie, light, temperature, etc.) also affect callus formation. Once established, callus cultures can be used to initiate regeneration of shoots. See, eg, Gurel et al. (2001) Turk J. Bot. 25:25-33; Dodds et al. (1995). Experiments in Plant Tissue Culture, Cambridge University Press; Gamborg (1995) Plant Cell, Tissue and Organ Culture, eds. G. Phillips and US Patent Application 20030180952, all incorporated herein by reference.

It is further appreciated that seed size and/or weight can be increased by increasing the growth rate of the seed or increasing its vigor. In addition, modulating plant tolerance to stress and modulating root, shoot and leaf development can increase plant yield and vigor, as described below. The term "vigor" as used herein refers to the relative condition, yield and growth rate of plants and/or specific plant parts, which may be reflected in various developmental characteristics including, but not limited to, chlorophyll concentration, rate of photosynthesis, total biomass , root biomass, grain quality and/or grain yield. Particularly in maize, vigor also reflects ear growth rate, ear volume, and/or silk elongation and expansion. Vigor relates to the ability of a plant to grow rapidly during early developmental stages and the successful establishment of a well-developed root system and well-developed photosynthetic organs after germination. Viability can be measured against different phenotypes under the same environmental conditions, or against the same or different genotypes under different environmental conditions.

Accordingly, the present invention further provides plants having modulated shoot and/or leaf development compared to control plants. In certain embodiments, plants of the invention have increased levels/activity of an IPT polypeptide of the invention. In other embodiments, plants of the invention have reduced levels/activity of IPT polypeptides of the invention.

VI. Regulation of reproductive tissue development

Methods of modulating reproductive tissue development are provided. In one embodiment, a method of modulating floral development of a plant is provided. "Modulating floral development" refers to any alteration in the structure of reproductive tissue of a plant compared to a control plant or plant part. "Modulating floral development" further includes any alteration in the reproductive tissue of a plant during development (ie delaying or accelerating floral development) compared to a control plant or plant part. Macroscopic changes include changes in reproductive organ volume, morphology, number or location, the developmental time it takes to form these structures, or the ability to maintain or proceed with the flowering process under environmental stress. Microscopic changes include changes in the type and morphology of the cells that make up the reproductive organs.

Methods of modulating floral development in plants include modulating (increasing or decreasing) the level and/or activity of an IPT polypeptide in a plant. In one method, an IPT polypeptide of the invention is provided. The IPT nucleotide sequence can be provided by introducing a polynucleotide containing the IPT nucleotide sequence of the present invention into a plant, expressing the IPT sequence, and regulating flower development. In certain embodiments, the IPT nucleotide construct introduced into the plant is stably integrated into the plant genome.

As mentioned above, the skilled person will know to use suitable promoters to regulate floral development in plants. Exemplary promoters of this technique include constitutive promoters, inducible promoters, shoot-biased promoters, and inflorescence-biased promoters (including female plant developing inflorescence-biased promoters), including those described elsewhere herein. Promoters listed in place.

In particular methods, floral development is modulated by increasing the level and/or activity of an IPT sequence of the invention. This method comprises introducing into a plant an IPT nucleotide sequence and increasing the activity of an IPT polypeptide. In certain methods, the IPT nucleotide construct introduced into a plant is stably integrated into the plant genome. An increase in the level and/or activity of an IPT sequence can result in one or more changes in floral development, including, but not limited to, accelerated flowering, increased number of flowers, and increased seed fixation compared to control plants. In addition, an increase in the level or activity of IPT sequences can inhibit flower senescence and alter the number of embryos in grains. See Young et al. (2004) Plant J. 38:910-22. Methods for determining such developmental changes in floral development are known in the art. See, e.g., Mouradov et al. (2002) The Plant Cell S111-S130, incorporated herein by reference.

In other methods, floral development is modulated by reducing the level and/or activity of an IPT sequence of the invention. Decreased levels and/or activity of IPT sequences can lead to grain abortion and sterile female plant inflorescences. Inducing delayed flowering or inhibiting flowering can be used to enhance the yield of forage crops such as alfalfa.

Accordingly, the present invention further provides plants having modulated floral development compared to floral development of control plants. Components include plants comprising reduced levels/activity of an IPT polypeptide of the invention and altered floral development. Components also include plants having increased levels/activity of an IPT polypeptide of the invention, wherein the plant can maintain or proceed with the flowering process during periods of stress.

VII. Regulation of stress tolerance in plants

Methods of altering the tolerance of plants to abiotic stress using the IPT sequences of the invention are provided. Increased seedling growth or early activity is often associated with increased stress tolerance. For example, rapid development of the root system of seedlings, including post-emergence of seedlings, is critical for survival under adverse conditions such as drought. Promoters for use in the present methods are described elsewhere herein and include low level constitutive, inducible or root-biased promoters - eg, root-biased promoters derived from the ZmlPT4 and ZmlPT5 regulatory sequences. Thus, in one method of the invention, tolerance to stress in plants can be increased or maintained by reducing the level of IPT activity in germinated seedlings compared to control plants. In other methods, the IPT nucleotide sequence is provided by introducing a polynucleotide containing the IPT nucleotide sequence of the present invention into the plant, expressing the IPT sequence, thereby increasing the tolerance of the plant to stress. In other embodiments, the IPT nucleotide construct introduced into the plant is stably integrated in the plant genome.

Also provided are methods of increasing or maintaining seed fixation during periods of abiotic stress. During stress periods (eg drought, salt, heavy metals, temperature, etc.), embryo development is usually aborted. In maize, interrupted embryo development can lead to mid-ear kernel abortion (Cheikh and Jones (1994) Plant Physiol. 106:45-51; Dietrich et al. (1995) Plant Physiol Biochem 33:327-336). Inhibition of this grain abortion maintains yield. Thus, methods of increasing stress tolerance in plants (eg, during flowering and seed development) are provided. Increased expression of the IPT sequences of the invention can regulate flower development during the stress phase, thus providing a method for maintaining or improving the flowering process of plants under stress. The method comprises increasing the level and/or activity of an IPT sequence of the invention. In one method, an IPT nucleotide sequence is introduced into a plant to increase the level and/or activity of the IPT polypeptide, thereby maintaining or improving the plant's tolerance to stress conditions. In other methods, an IPT nucleotide construct introduced into a plant is stably integrated into the plant genome. See for example WO 00/63401.

Unfavorable environments can lead to significant yield instability during the lag phase of seed development. At this stage, the seeds undergo drastic changes in ultrastructure, biochemistry, and sensitivity to environmental disturbances, but only minor changes in dry weight accumulation occur. Two important events that occur during the lag phase are the initiation and division of endosperm cells and amyloplasts (which are the sites of starch precipitation). It has been shown that during the lag phase (10-12 days post-pollination (DAP) in maize), cytokinin concentrations increase sharply before the maximum rate of endosperm cell division and amyloplast formation, suggesting a central role for this hormone in these processes , known as the "pool strength" of the developing seed. Cytokinins have been shown to play an important role in determining seed size, reducing apical abortion and increasing seed fixation under adverse environmental conditions. For example, increasing temperature can affect seed formation. Elevated temperature can inhibit the accumulation of cytokinins, reduce the separation of germ cells and the number of amyloplasts, thereby increasing the abortion of grains.

Seed sink capacity in maize plays a major role in the formation of endosperm cells and starch grain numbers during the first 6 to 12 DAP. The number of endosperm cells and amyloplasts formed is highly correlated with final grain weight (Capitanio et al., 1983; Reddy and Daynard, 1983; Jones et al., 1985, 1996; Engelen-Eigles et al., 2000). Hormones, especially cytokinins, stimulate cell division, plastid formation and other processes important in establishing grain pool capacity (Davies, 1987). Cytokinin levels can be regulated, for example, with the ZmlPT2 promoter that drives expression of the Agrobacterium IPT gene. Likewise, endosperm and/or stalk-biased promoters can be used to increase the level and/or duration of ZmlPT2 expression, thereby increasing cytokinin levels, which in turn increases pool strength and grain yield. Capitano, R., Gentinetta, E. and Motto, M. (1983). Grain weight and its components in maize inbredlines. Maydica 23:365-379. Jones, R.J., Roessler, J. and Ouattar, S. (1985). Thermal environment during endosperm cell division in maize: effects on number of endosperm cells and starch granules. Crop Science 25: 830-834. Jones, R.J., Schreiber, B.M.N. and Roessler, J. (1996). Kernel sink strength capacity in maize: Genotypic and maternal regulation. Crop Science 36:301-306. Davies, G.C. (1987). The planthormones: their nature, occurrences and function. P 1-12. In P.J. Davies and M. Nijhoff (ed.). Plant hormones and their role in plant growth and development. Dordrecht, the Netherlands. Engelen-Eigles G., Jones, R.J. and Phillips R.L. (2000). DNA endoreduplication in maizeendosperm cells: the effect of exposure to short-term high temperature. Plant, Cell and Environment 23: 657-663.

Thus provided are methods of increasing IPT activity and/or levels in developing inflorescences, thereby increasing levels of cytokinins, giving seeds developing in unfavorable environments their full potential in terms of volume, reduced grain abortion, and buffered seed settling. genetic potential. These methods further enable plants to maintain and/or improve flowering in adverse environments.

In this technical solution, various promoters can be used to direct the expression of sequences that can increase the level and/or activity of IPT polypeptides, including but not limited to, constitutive promoters, seed-biased promoters, developmental seed or grain Grain promoters, meristem-biased promoters, stress-inducible promoters, and inflorescence-biased promoters (eg, gynecologically developing inflorescence promoters). In one approach, a promoter that is stress-insensitive and expresses in tissues of developing seeds during the lag phase is used. "Stress insensitive" means that the expression level of the sequence operably linked to the promoter does not change or changes only slightly under stress conditions. A "lag phase" promoter is a promoter that is active during the lag phase of seed development. Such developmental periods are described elsewhere herein. "Developing seed bias" refers to a promoter that enhances the expression of IPT in developing seeds. These stress-insensitive promoters expressed in tissues of developing seeds during developmental delay are known in the art, including Zag2.1 (Theissen et al. (1995) Gene 156:155-166, Genbank Accession No. X80206) and mzE40 (Zm40) (US Patent 6,403,862 and WO01/2178).

The expression construct may further contain a nucleotide sequence encoding a peptide signal sequence that alters the level and/or activity of a cytokinin in the mitochondria or chloroplast. See, e.g., Neupert (1997) Annual Rev. Biochem. 66:863-917; Glaser et al. (1998) Plant Molecular Biology 38:311-338; Duby et al. (2001) The Plant J 27(6):539-549 .

Methods for determining increased seed fixation under abiotic stress are known in the art. For example, plants with increased IPT activity can be monitored under various stress conditions and compared to control plants. For example, plants with increased cytokinin synthesis activity can withstand various degrees of stress during the anthesis and seed fixation stages. Genetically altered plants with increased cytokinin synthesis activity have a greater number of developing grains than control plants under the same conditions.

Thus, the present invention further provides plants capable of increasing or maintaining yield and/or increasing or maintaining the flowering process during periods of abiotic stress (drought, salt, heavy metals, temperature extremes, etc.). In certain embodiments, plants that increase or maintain yield under abiotic stress have increased levels/activity of an IPT polypeptide of the invention. In certain embodiments, the plant contains an IPT nucleotide sequence of the invention operably linked to a promoter that initiates expression in the plant cell. In certain technical schemes, the nucleic acid molecule containing the IPT nucleotide sequence of the present invention operably linked to a promoter that promotes expression in plant cells is stably integrated in the genome of such plants.

VIII. Methods of Using IPT Promoter Polynucleotides

The IPT promoter-containing polynucleotides of the present invention and variants and fragments thereof, when combined with a DNA construct such that the promoter sequence is operably linked to a nucleotide sequence containing a polynucleotide of interest, can be used in Genetic manipulation of any host cell, preferably a plant cell. In this manner, the IPT promoter polynucleotide of the invention is provided in an expression cassette together with a heterologous polynucleotide sequence of interest for expression in the host cell of interest. As described in Example 2 below, the IPT promoter sequence of the present invention is expressed in various tissues, so that the promoter sequence can be used to regulate the temporal or spatial expression of the target polynucleotide.

Synthetic hybrid promoter regions are known in the art. Such a region includes an upstream promoter element of a polynucleotide operably linked to a promoter element of another polynucleotide. In one technical solution of the present invention, expression of heterologous sequences is regulated by a synthetic hybrid promoter containing the IPT promoter sequence of the present invention or a variant or fragment thereof operably linked to upstream promoter elements of the heterologous promoter. Upstream promoter elements involved in plant defense systems have been identified and can be used to generate synthetic promoters. See, eg, Rushton et al. (1998) Curr. Opin. Plant Biol. 1:311-315. Alternatively, the synthetic IPT promoter sequence may contain copies of the upstream promoter elements found in the IPT promoter sequence.

It is understood that the promoter sequences of the present invention can be used with native IPT coding sequences. DNA constructs containing an IPT promoter operably linked to its native IPT gene can be used to transform any plant of interest to obtain desired phenotypic changes—for example, modulation of cytokinin levels, modulation of root, shoot, leaf, flower, and embryo development , stress tolerance and any other phenotype described herein.

The promoter nucleotide sequences and methods described herein can regulate the expression of any heterologous nucleotide sequence in a host plant, thereby altering the phenotype of the plant. Various changes in phenotype are worthy of attention, including modulation of fatty acid composition in plants, changes in plant amino acid content, changes in plant pathogen defense mechanisms, etc. By providing expression of heterologous products or increasing expression of endogenous products in plants. Alternatively, these results may be achieved by reducing the expression of one or more endogenous products, in particular enzymes or cofactors, in the plant. These changes can result in altered phenotypes in transformed plants.

Target genes are a reflection of the commercial market and its interests involved in crop development. New crops and technologies will emerge as target crops and markets change and developing countries open up to world markets. In addition, as our understanding of agronomic traits and traits such as yield and heterosis increases, the selection of genes for transformation will change. Typical classes of target genes include, for example, genes involved in information such as zinc fingers, genes involved in communication such as kinases, and genes involved in housekeeping such as heat shock proteins. More specific categories of transgenes include, for example, genes encoding agronomically important traits, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest typically include those involved in oil, starch, carbohydrate or nutrient metabolism and those affecting grain size, sucrose content, etc.

In one embodiment, the target sequence improves plant growth and/or crop yield. In a more specific technical solution, the expression of the target nucleotide sequence improves the plant's response to the stress induced under high-density growth conditions. For example, sequences of interest include agronomically important genes that improve primary or lateral root systems. These genes include, but are not limited to, nutrient/water delivery systems and growth inducers. Examples of these genes include, but are not limited to, maize plasma membrane H ⁺ -ATPase (MHA2) (Frias et al. (1996) Plant Cell 8: 1533-44); AKT1, a component of the potassium uptake system in Arabidopsis ( Spalding et al. (1999) J GenPhysiol 113: 909-18); activate the RML gene (Cheng et al. (1995) Plant Physiol 108: 881) of the cell division cycle in root apical cells; maize glutamate synthase gene ( Sukanya et al. (1994) Plant Mol Biol 26: 1935-46) and hemoglobin (Duff et al. (1997) J. Biol. Chem 27: 16749-16752, Arredondo-Peter et al. (1997) Plant Physiol. 115: 1259-1266; Arredondo-Peter et al. (1997) Plant Physiol 114:493-500, and references cited therein). Target sequences are also used to express antisense nucleotide sequences to genes that can negatively affect root development.

In addition, agronomically important traits such as oil, starch and protein content can additionally be genetically altered by traditional breeding methods. Modifications included increased oleic acid content, changes in the ratio of saturated and unsaturated oils, increased levels of lysine and sulfur, provision of essential amino acids, and modification of starch. Modifications of the Hordothionin protein are described in US Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, incorporated herein by reference. Another example is the lysine and/or sulfur-enriched seed protein encoded by soybean 2S albumin described in U.S. Patent 5,850,016, and in Williamson et al. (1987) Eur.J.Biochem.165:99-106 Chymotrypsin inhibitors in barley as described in , incorporated herein by reference.

Derivatives of coding sequences can be prepared by site-directed mutagenesis to increase the levels of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. Patent Application 08/740,682, filed November 1, 1996, and WO 98/20133, the inventions of which are incorporated by reference Introduced in this article. Other proteins include those from sunflower seeds (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Illinois), pp. 497-502; as incorporated herein by reference); maize (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; incorporated herein by reference) and rice (Musumura et al. (1989) Plant Mol. Biol. 12: 123, incorporated herein by reference) is a methionine-enriched plant protein. Other agronomically important genes encode milk, Floury 2, growth factors, seed storage factors and transcription factors.

Insect resistance genes encode resistance to pests such as carnivores, caterpillars, European corn borer, etc. that greatly reduce yields. These genes include, for example, Bacillus thuringiensis toxic protein genes (US Patents 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881 and Geiser et al. (1986) Gene 48:109) and the like.

Genes encoding disease-resistant properties include detoxification genes, such as anti-fumonosin (U.S. Patent No. 5,792,931); avirulence (avr) and disease-resistance (R) genes (Jones et al. (1994) Science 266: 789; Martin et al. ( 1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089) et al.

Genes encoding disease resistance traits include genes such as antifumonosin (US Patent 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262: 1432 and Mindrinos et al. (1994) Cell 78: 1089) and other detoxification genes.

Herbicide resistance traits include those encoding acetolactate synthase (ALS), particularly sulfonylurea-type herbicides (e.g., acetolactate synthase containing mutations leading to such resistance, particularly S4 and/or Hra mutations ( ALS) gene) action herbicide resistance gene, encoding such as glufosinate or glufosinate (i.e. bar gene) the herbicide resistance gene that inhibits the action of glutamate synthase, or other such herbicide resistance gene known in the art gene-like. The Bar gene encodes resistance to the herbicide glufosinate, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene mutants encode resistance to the sulfonylurea herbicide.

A sterility gene is also encoded in the expression cassette, providing selection for physiological emasculation. Examples of genes used in these methods include genes for male plant tissue preference and genes for male plant sterility such as DAM described in US Pat. Nos. 5,750,868; 5,689,051 and 6,281,348. Other genes include kinases and genes encoding compounds that are toxic to male or female gametophyte development.

Grain quality is reflected by such traits as level and type of oil, saturation and unsaturation, quality and quantity of essential amino acids, ie level of cellulose. Modified hordothionin proteins in maize are described in US Pat.

Genes may also be used to encode commercial traits, either to increase starch in, for example, ethanol production, or to provide protein expression. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in US Patent 5,602,321. Genes such as β-ketothiolase, PHBase (polyhydroxybutyrate synthase) and acetyl-CoA reductase (in terms of Schubert et al. (1988) J. Bacteriol. 170:5837-5847) favor polyhydroxybutyrate (PHAs) expression.

Exogenous products include enzymes and products of plants and other products of prokaryotic and other eukaryotic origin. These products include enzymes, cofactors, hormones, etc. The level of proteins, especially modified proteins with improved amino acid profile to increase the nutritional value of the plant, can be increased. This can be achieved by expressing such proteins with enhanced amino acid content.

IX. Antibody production and use

Antibodies can be raised against native and recombinant forms of the proteins of the invention, including variants and fragments thereof. Various methods of preparing antibodies are known to those skilled in the art. Various analytical methods can be used to produce the highly hydrophilic properties of the proteins of the invention. These methods can be used to guide the skilled person to screen the peptides of the present invention, so as to generate or screen antibodies that specifically react with the proteins of the present invention under immunization conditions. See e.g. J. Janin, Nature, 277 (1979) 491-492; Wolfenden, et al., Biochemistry 20 (1981) 849-855; Kyte and Doolite, J. Mol Biol. 157 (1982) 105-132; Rose, et al., Science 229(1985) 834-838. Antibodies can be used to screen the expression library of specific expression products such as normal or abnormal proteins, or to change their levels, so as to detect or diagnose various diseases related to the corresponding antigens. For example, an assay that indicates high levels of an IPT protein of the invention can be used to detect plants or specific plant parts with elevated cytokinin levels. Typically antibodies in such procedures are labeled with moieties that are readily detected in the presence of antigen/antibody binding.

A general description of the technique follows; however, the skilled artisan will appreciate that modifications are known according to the known methods described below.

Various immunogens can be used to generate antibodies specifically reactive with the proteins of the invention. Polypeptides encoded by isolated recombinant, synthetic or natural polynucleotides of the invention are preferred antigens for raising monoclonal or polyclonal antibodies. The polypeptides of the invention may optionally be denatured or cleaved prior to injection into antibody-producing animals. Monoclonal or polyclonal antibodies can be generated for use in the immunoassays described below to determine the presence and amount of the protein of the invention. Methods of producing polyclonal antibodies are known to those skilled in the art. Briefly, an antigen, preferably a purified protein, protein coupled to a suitable carrier (e.g. GST, keyhole limpet hemocyanin, etc.) or integrated into an immunization vector such as a recombinant vaccinia virus (see U.S. Patent No. 4,722,848) The protein is mixed with an adjuvant, and the mixture is used to immunize animals. The animal's immune response to the immunogen product is monitored by collecting experimental blood and detecting the reaction titer to the target protein. When suitably high antibody titers to the immunogen are obtained, the animal is bled and antiserum is prepared. Specific monoclonal and polyclonal antibodies typically have antibody binding to their cognate monovalent antigen with an affinity constant of at least 10 ⁶ -10 ⁷ , usually at least 10 ⁸ , 10 ⁹ , 10 ¹⁰ , or higher than 10 ¹¹ L/mole location. If desired, further fragmentation of the antiserum can enrich for antibodies reactive with the protein (see, e.g., Coligan, Current Protocols in Immunology, Wiley/Greene, NY (1991) and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY (1989)).

Antibodies to predicted fragments of the invention, including binding fragments and single-chain recombinant portions thereof, such as conjugates of fragments linked to carrier proteins as described above, can be produced by immunizing animals. Typically, the immunogen of interest is a protein of at least 5 amino acids, more typically 10 amino acids in length, usually 15 to 20 amino acids in length, but can be longer. Peptides are typically coupled to carrier proteins (eg, as fusion proteins) or expressed recombinantly in immunological vectors. The epitope on the peptide to which the antibody binds is typically 3 to 10 amino acids in length.

Monoclonal antibodies are prepared from hybridoma cells secreting the desired antibody. Monoclonal antibodies are screened for binding to the protein from which the antigen is derived. In, for example, Basic and Clinical Immunology, 4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos, CA and references cited therein, Harlow and Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice, 2nd ed. Instructions or techniques for preparing monoclonal antibodies can be found in , Academic Press, New York, NY (1986) and Kohler and Milstein, Nature 256:495-497 (1975). Briefly, the method is carried out by injecting an animal with an antigen comprising a protein of the invention. The animals were then killed and cells collected from their spleens were fused with myeloma cells. The result is a hybrid cell or "hybridoma" that can propagate in vitro. The population of hybridomas is then screened to isolate single clones, each of which secretes a single antibody species directed against the antigen. In this manner, the individual antibody species obtained are the product of infinitely differentiated, clonal individual B cells produced by the animal in response to specific sites recognizable on the antigenic material.

Other suitable techniques include screening recombinant antibody libraries of phage or the same vector (see for example Huse et al., Science 246:1275-1281 (1989) and Ward, et al., Nature 341:544-546 (1989) and Vaughan et al. ., Nature Biotechnology, 14:309-314 (1996)). Moreover, recombinant immunoglobulins can be produced. See Cabilly, US Patent 4,816,567 and Queen et al., Proc. Nat'l Acad. Sci. 86:10029-10033 (1989).

Antibodies against polypeptides of the invention are also used in affinity chromatography to isolate proteins of the invention. Using, for example, a solid support to which antibodies can be attached - eg particles such as Sepharose, SEPHADEX, etc. - to prepare columns, cell lysates are passed through the columns, rinsed, and treated with increasing concentrations of mild denaturants to release the purified protein.

Usually, the proteins and antibodies of the present invention are labeled with a substance providing a detectable signal by covalent or non-covalent linkage. Various labeling and conjugation techniques are known and widely reported in the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent molecules, chemoimmunogroups, magnetic beads, and the like.

protein immunoassay

The method of detecting the proteins of the invention is not a critical aspect of the invention. In certain embodiments, proteins are detected and/or quantified using any of a variety of known immunological binding assays (see, eg, US Pat. Nos. 4,366,241; 4,376,110; 4,517,288 and 4,837,168). For a review of immunoassays, see Methods in Cell Biology, Vol.37: Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds. (1991). In addition, the immunoassay of the present invention can be used in any form, such as Enzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Florida (1980); Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers B.V., Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, FL (1987); Principles and Practice of Immunoassaysm, Price and Newman Eds., Stockton Press, NY (1991) and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY (1988).

Immunobinding assays (or immunoassays) typically use "capture reagents" that specifically bind to and generally immobilize the analyte (in this case the protein of the invention). Capture reagents are components that specifically bind to an analyte. In a specific technical solution, the capture reagent is an antibody that specifically binds to the protein of the present invention. Antibodies can be produced by any method known to those of skill described herein.

Immunoassays also typically utilize labels that specifically bind to and label the bound complex formed by the capture reagent and the analyte. Such a label may itself be a component of an antibody/analyte-containing complex. Accordingly, the marker may be a labeled protein of the present invention or a labeled antibody specifically reactive with a protein of the present invention. Alternatively, the label may be a third component - eg another antibody that specifically binds to the antibody/protein complex.

In an assay, incubation and/or washing steps are required after every reagent binding. The incubation step can range from 5 seconds to several hours, typically 5 minutes to 24 hours. However, the incubation time depends on the detection method, analyte, solution volume, concentration, etc. Although reaction temperatures can range over a wide range—for example, 10°C to 40°C—assays are usually performed at room temperature.

The details of the immunoassay of the present invention vary depending on the particular assay used. Methods for detecting the protein of the present invention in a biological sample generally involve contacting the biological sample with an antibody that specifically reacts with the protein of the present invention under immunoreactive conditions. Thus, the antibody is bound to the protein under immunoreactive conditions, and the presence of the bound antibody can be detected directly or indirectly.

A. Non-competitive detection method

Immunoassays for detecting proteins of the invention include competitive and non-competitive formats. Non-competitive immunoassays are assays that directly measure the amount of captured analyte (ie, protein of the invention). In one embodiment, a "sandwich" assay, a capture reagent (eg, an antibody that specifically reacts with a protein of the invention under immunoreactive conditions) can be directly bound to the solid support on which it is immobilized. This immobilized antibody captures proteins present in the test sample. In this way, the immobilized protein is bound to a label—such as a labeled secondary antibody. Alternatively, the second antibody may be unlabeled, but instead be bound by a labeled third antibody specific for the antibody species from which the second antibody was derived. The second antibody can be modified with a detectable substance such as biotin to specifically bind to a third labeled molecule such as enzyme-labeled streptavidin.

B. Competition detection method

In a competitive assay format, the amount of analyte present in the sample is determined by determining the amount of the added (exogenous) analyte (e.g. a protein of the invention) that is absorbed by the analyte present in the sample from a capture reagent (e.g. The amount of substitution (or competition) by the antibody with specific reaction) is determined indirectly. In a competition assay, a known amount of analyte is added to a sample, and the sample is contacted with a capture reagent that specifically binds a protein of the invention. The amount of protein bound to the capture reagent is inversely proportional to the concentration of analyte present in the sample.

In one embodiment, the antibody is immobilized on a solid support. The amount of protein bound to the antibody can be determined by determining the amount of protein present in the protein/antibody complex, or alternatively, the amount of protein remaining unbound. The amount of protein can be detected by labeling the protein.

The hapten inhibition assay is another competition assay. In this assay, a known analyte, such as a protein of the invention, is immobilized on a solid support. Known amounts of antibodies that specifically react with the protein under immunoreactive conditions are added to the sample, and the sample is then contacted with the immobilized protein. In this case, the amount of antibody bound to the immobilized protein is inversely proportional to the amount of protein present in the sample. Furthermore, the amount of antibody immobilized can be determined by detecting the immobilized portion of the antibody or the remaining portion of the antibody in solution. As noted above, direct detection can be performed, wherein the antibody is labeled, or indirect detection by the subsequent addition of a label that specifically binds to the antibody.

C. Generation of Pooled Antisera for Immunoassays

Proteins that specifically bind to or specifically immunoreact with antibodies raised against a defined antigen can be detected in immunoassays. Immunoassays use polyclonal antisera directed against polypeptides of the invention (ie, antigenic polypeptides). The antiserum is chosen to have low cross-reactivity with other proteins, and any such cross-reactivity is removed by immunoabsorption prior to application of the immunoassay (e.g., with proteins of different substrate specificity (e.g., different enzymes). ) and/or antisera with the same substrate specificity but different forms of protein for immunoabsorption).

To generate antisera for use in immunoassays, the polypeptides of the invention are isolated as previously described. For example, recombinant proteins are produced in mammalian or other eukaryotic cell lines. Inbred strains of mice are immunized with protein using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra). Alternatively, synthetic polypeptides derived from the inventive sequences and coupled to carrier proteins can be used as immunogens. Polyclonal sera are collected and the immunogenic polypeptides are titrated by immunoassay, eg, with a solid phase immunoassay immobilized on a solid support. Screen polyclonal antisera with a titer of ¹⁰⁴ or higher and test for polypeptides specific for different types or substrates by competitive binding immunoassays such as those described in Harlow and Lane 570-573 above cross-reactivity. Preferably, two or more different types of polypeptides are used in the assay. These different types of polypeptides can be used as competitors to identify antibodies that specifically bind to the test polypeptide. Competing polypeptides can be produced as recombinant proteins and isolated by standard molecular biology methods and protein chemistry techniques as described herein.

Immunoassays in a competitive binding format can be used to measure cross-reactivity. For example, the immunizing polypeptide is immobilized on a solid support. The added protein competes with the immobilized antigen for binding to the antiserum. The ability of the above proteins to compete with the immobilized protein for binding to the antiserum was compared with that of the immune polypeptide. The percent cross-reactivity of the above proteins was calculated by standard methods. Those antisera with less than 10% cross-reactivity to different types of polypeptides were screened and pooled. Antibodies with cross-reactivity are then removed from the pooled antisera by immunoabsorption of different types of polypeptides.

The immunoabsorbed and pooled antisera are then used in the competitive binding immunoassay to compare against a second "target" polypeptide against the immunized polypeptide. For comparison, both polypeptides were tested over a wide range of concentrations and the amount of each polypeptide which inhibited 50% of the binding of the immobilized protein to the antiserum was determined by standard methods. The target polypeptide is considered to specifically bind to antibodies raised by the immunizing protein if the amount of target polypeptide required is less than twice the amount of immunizing polypeptide required. For the final determination of specificity, the pooled antisera are subjected to an extensive immunouptake with the immunizing polypeptide until no binding to the used polypeptide is detectable in the immunouptake. The fully immunoabsorbed antiserum is then tested for reactivity with the polypeptide to be tested. If there is no reactivity, the peptide to be tested specifically binds to the antiserum raised by the immune protein.

D. Other detection methods

In a particular protocol, Western blot (immunoblot) analysis is used to detect and quantify the protein of the invention present in the sample. The technique generally involves separating sample proteins by gel electrophoresis based on molecular weight, transferring the separated proteins to a suitable solid support (such as nitrocellulose, nylon, or derivatized nylon filters), and separating the samples with and Antibodies specifically binding to the protein of the present invention are incubated together. Antibodies bind specifically to proteins on solid supports. These antibodies can be directly labeled or detected using a labeled antibody that specifically binds these antibodies (eg, labeled goat anti-mouse antibody).

E. Protein quantification

The protein of the present invention can be detected and quantified by any of various methods known to those skilled in the art. Including analytical biochemical methods - such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, etc., and various immunological methods - such as liquid phase or gel precipitants Reaction, immunodiffusion (one-way or two-way), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence, etc.

F. Reduce non-specific binding

The skilled artisan will understand that in immunoassays and analyte purification there is often a need to reduce non-specific binding. Where the assay involves an antigen, antibody or other capture reagent immobilized on a support, it is desirable to reduce the amount of non-specific binding to the support. Methods of reducing this non-specific binding are known to those skilled in the art. Typically, this involves coating the support with similar protein components. In particular, protein components such as bovine serum albumin (BSA), skim milk and gelatin are commonly used.

G. Labeling of immunoassays

Labeling reagents can be, for example, monoclonal antibodies, polyclonal antibodies, binding proteins and complexes, or polymers such as affinity matrices, carbohydrates or fats. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Detection can be performed by any known method - e.g. immunoblotting, Western analysis, gel retardation, fluorescence in situ hybridization (FISH), tracking with radioactive or bioluminescent markers, NMR, electron paramagnetic resonance, stop Flow spectroscopy, column chromatography, capillary electrophoresis, or other methods of tracking molecules based on changes in volume and/or charge. The particular label or detection group used in the detection is not a critical aspect of the invention. The detection moiety can be any substance with detectable physical or chemical properties, including magnetic beads, fluorescent dyes, radioactive labels, enzymes and colorimetric labels or colored glass or plastic beads, such as the nucleic acid labels discussed above. Labels can be coupled directly or indirectly to the analyte according to methods known in the art. As noted above, a variety of labels can be used, and the choice of label depends on the desired sensitivity, ease of coupling to the compound, stability requirements, available instrumentation, and handling protocols. Means of detecting markers are known to those skilled in the art.

Non-radioactive labels are usually attached by indirect means. Typically, a ligand molecule (eg, biotin) is covalently bound to the molecule. The ligand is then bound to an anti-ligand molecule (eg streptavidin) which is itself detectable or covalently bound to a signaling system such as a detection enzyme, fluorescent compound or chemiluminescent compound. A variety of ligands and anti-ligands can be used.

Molecules can also be coupled directly to signal-generating compounds, such as enzymes or fluorophores. The target enzymes used as markers are mainly hydrolases, especially phosphatases, esterases and glycosidases, or oxidoreductases, especially peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, dispersone, etc. Chemiluminescent compounds include luciferins, 2,3-dihydrophthalazinediones, such as luminol. A review of the various marker or signal generating systems available can be found in US Patent 4,391,904, incorporated herein by reference.

Certain assay formats do not require the use of labeled components. For example, the presence of the target antibody can be detected using an agglutination assay. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this way, there is no need to label any components, and the presence of the target antibody can be detected by simple visual inspection.

Detection of compounds that modulate enzyme activity or expression

A catalytically active polypeptide of the invention can be contacted with a compound to determine whether the compound binds and/or modulates the enzymatic activity of such polypeptide. The polypeptide used has at least 20%, 30%, 40%, 50%, 60%, 70% or 80% of the specific activity of the native, full-length enzyme of the invention. Generally, the polypeptide is in a range sufficient to determine the effect of the compound, typically about 1 nM to 10 μM. Again, the concentration of the compound to be tested is from 1 nM to 10 μM. The skilled artisan will appreciate that a variety of factors - such as enzyme concentration, ligand concentration (i.e., substrate, product, inhibitor, activator), pH, ionic strength, and temperature - need to be controlled to obtain useful kinetic data and determine binding Or whether there is a compound that modulates the activity of the polypeptide. Methods for measuring enzyme kinetics are known in the art. See, eg, Segel, Biochemical Calculations, 2 ^nd ed., John Wiley and Sons, New York (1976).

Technical scheme of the present invention comprises as follows:

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence comprising SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 ;

(b) containing at least an amino acid sequence with 85% sequence identity, wherein said polypeptide has cytokinin synthesis activity;

(c) composed under stringent conditions with SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28 , 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 complementary sequence hybridization The amino acid sequence encoded by the nucleotide sequence, wherein the stringent conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C, washing in 0.1X SSC at 60°C to 65°C, wherein the The polypeptide has cytokinin synthesis activity; and

(d) Contains at least 50 of SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 An amino acid sequence of consecutive amino acids, wherein the polypeptide has cytokinin synthesis activity.

2. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, the nucleotide sequence of 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76;

(b) encoding an amino acid comprising SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 the nucleotide sequence of the sequence;

(c) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42 , 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 having at least 85% sequence identity a nucleotide sequence, wherein the polynucleotide encodes a polypeptide having cytokinin synthesis activity;

(d) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, At least 50 of 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 or their complementary sequences the nucleotide sequence of the nucleotides; and

(e) a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence in a) under stringent conditions, wherein the stringent conditions include hybridization at 37°C in 50% formamide, 1M NaCl, 1% SDS, at 60 °C to 65 °C rinse in 0.1X SSC.

3. An expression cassette comprising the polynucleotide in technical scheme 2.

4. The expression cassette according to technical solution 3, wherein the polynucleotide is linked to a promoter that drives expression in plants.

5. A plant containing a polynucleotide linked to a promoter that initiates expression in a plant, wherein said polynucleotide contains a nucleotide sequence selected from the group consisting of:

(a) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, the nucleotide sequence of 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76;

(b) encoding an amino acid comprising SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 the nucleotide sequence of the sequence;

(c) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42 , 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 having at least 85% sequence identity a nucleotide sequence, wherein the polynucleotide encodes a polypeptide having cytokinin synthesis activity;

(d) a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence in a) under stringent conditions, wherein the stringent conditions include hybridization at 37°C in 50% formamide, 1M NaCl, 1% SDS, at 60 Washing in 0.1X SSC at °C to 65°C, wherein the polynucleotide encodes a polypeptide having cytokinin synthesis activity; and

(e) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 or their complementary sequences at least 50 contiguous Nucleotide sequence of nucleotides.

6. The plant according to technical solution 5, wherein said plant has a regulated cytokinin level.

7. The plant according to technical solution 6, wherein the cytokinin level is regulated in vegetative tissue, reproductive tissue, or vegetative tissue and reproductive tissue.

8. The plant according to claim 6, wherein the cytokinin level is increased.

9. The plant according to technical scheme 6, wherein the cytokinin level is reduced.

10. The plant according to item 5, 6, 7, 8 or 9, wherein the promoter is a tissue-biased promoter, a constitutive promoter or an inducible promoter.

11. The plant according to technical solution 10, wherein the promoter is a root-biased promoter, a leaf-biased promoter, a branch-biased promoter or an inflorescence-biased promoter.

12. The plant according to technical solution 5, wherein the plant has regulated flower development.

13. The plant of technical scheme 5, wherein said plant has regulated root development.

14. The plant of claim 13, wherein the modulated root development comprises at least one increase in root growth or increase in lateral or adventitious root formation.

15. The plant according to claim 5, wherein the plant has an altered ratio of striped roots.

16. The plant according to technical scheme 5, wherein said plant has increased seed size or increased seed weight.

17. The plant of technical scheme 16, wherein the increase in seed size or seed weight comprises an increase in at least one of embryo volume, embryo weight, cotyledon volume, or cotyledon weight.

18. The plant of technical scheme 5, wherein the vigor or biomass yield of the plant is increased.

19. The plant according to technical solution 5, wherein the stress tolerance of the plant is maintained or improved.

20. The plant according to technical scheme 19, wherein the size of the plant is increased or maintained.

21. The plant according to item 19, wherein the abortion at the top of the grain is reduced.

22. The plant according to claim 19, wherein seed fixation of said plant is increased or maintained.

23. The plant according to claim 19, 20, 21 or 22, wherein said promoter is stress-insensitive and is expressed in developing seed tissues and related maternal tissues at least during the lag phase of seed development.

24. The plant according to claim 5, wherein the growth of shoots of the plant is reduced.

25. The plant according to technical scheme 5, wherein said plant has delayed senescence or increased vegetative growth.

26. The plant according to any one of items 5 to 9, 12 to 22, 24 and 25, wherein the polynucleotide is stably integrated into the plant genome.

27. Transformed seeds of the plant of claim 26.

28. A plant genetically modified at a native genomic locus encoding a polypeptide selected from the group consisting of:

(a) an amino acid sequence comprising SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 ;

(b) containing SEQ ID NO: SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 amino acid sequences having at least 85% sequence identity, wherein said polypeptide has cytokinin synthesis activity;

wherein said plant is genetically modified to increase, decrease or eliminate the activity of said polypeptide.

29. The plant according to any one of items 5 to 9, 12 to 22, and 24 to 28, wherein said plant is a monocot.

30. The plant according to technical solution 29, wherein the monocotyledonous plant is corn, wheat, rice, barley, sorghum or rye.

31. The plant according to any one of items 5 to 9, 12 to 22, and 24 to 28, wherein said plant is a dicotyledonous plant.

32. A method for reducing or removing polypeptide activity in plants, comprising introducing into said plants a compound containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 , 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71 , 72, 73, 74 or 76, or a polynucleotide of a fragment of said fragment.

33. The method of technical scheme 32, wherein the polynucleotide can reduce the level of cytokinin in the plant.

34. The method of technical scheme 32, wherein the activity of the polypeptide is reduced or eliminated in vegetative tissue, reproductive tissue, or vegetative tissue and reproductive tissue.

35. The method according to technical solution 32, wherein the introduced polynucleotide is linked to a tissue-biased promoter, a constitutive promoter or an inducible promoter.

36. The method of technical scheme 35, wherein the promoter is a root-biased promoter.

37. The method in technical scheme 32, wherein reducing or removing the activity of the polypeptide can regulate the development of plant roots.

38. The method of technical scheme 37, wherein the regulated root development comprises at least one of root growth increase or lateral root or adventitious root formation increase.

39. A method of increasing the level of a polypeptide in a plant, comprising introducing into said plant a polynucleotide comprising a nucleotide selected from the group consisting of:

(a) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, the nucleotide sequence of 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76;

(b) encoding an amino acid comprising SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66 or 77 the nucleotide sequence of the sequence;

(c) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42 , 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 having at least 85% sequence identity a nucleotide sequence, wherein the polynucleotide encodes a polypeptide having cytokinin synthesis activity;

(d) containing SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, At least 40 of 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 or their complementary sequences the nucleotide sequence of the nucleotides; and

(e) a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence in a) under stringent conditions, wherein the stringent conditions include hybridization at 37°C in 50% formamide, 1M NaCl, 1% SDS, at 60 °C to 65 °C and rinsed in 0.1X SSC, wherein the polynucleotide encodes a polypeptide having cytokinin synthesis activity.

40. The method of technical scheme 39, wherein expressing the polynucleotide can increase the level of cytokinin in the plant.

41. The method in technical scheme 39 or 40, wherein the polypeptide level is increased in vegetative tissue, reproductive tissue or vegetative tissue and reproductive tissue.

42. The method according to technical scheme 39 or 40, wherein the promoter is a tissue-biased promoter, a constitutive promoter or an inducible promoter.

43. The method in technical scheme 42, wherein the promoter is a root-biased promoter, a leaf-biased promoter, a branch-biased promoter, a seed-biased promoter, or a grain-biased promoter or inflorescence-biased promoters.

44. The method according to technical scheme 39 or 40, wherein the stress tolerance of the plant is maintained or improved.

45. The method of technical scheme 44, wherein the plant size is increased or maintained.

46. The method of technical scheme 44, wherein seed abortion is reduced.

47. The method of technical scheme 44, wherein the seed fixation of the plant is increased or maintained.

48. The method according to technical scheme 44, wherein the promoter is insensitive to stress and is expressed in developing seed tissue during the developmental delay period.

49. The method in technical scheme 39 or 40, wherein increasing the level of the polypeptide can increase the shoot growth of the plant.

50. The method in technical scheme 39 or 40, wherein increasing the activity of the polypeptide can increase the seed size or seed weight of the plant.

51. The method of technical solution 50, wherein the increased seed size or seed weight comprises an increase in at least one of embryo volume, embryo weight, cotyledon volume or cotyledon weight.

52. The method in technical scheme 39 or 40, wherein increasing the activity of the polypeptide can increase the plant yield or plant vigor of the plant.

53. The method in technical scheme 39 or 40, wherein increasing the activity of the polypeptide can regulate the development of flowers.

54. The method in technical scheme 39 or 40, wherein increasing the polypeptide level can delay senescence or increase leaf growth.

55. The method according to any one of technical solutions 32-40, wherein the plant is a dicotyledonous plant.

56. The method according to any one of technical solutions 32-40, wherein the plant is a monocot.

57. The method of technical solution 56, wherein the monocot is corn, wheat, rice, barley, sorghum or rye.

58. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 25 or 75.

59. A DNA construct comprising a promoter linked to a target nucleotide, wherein the promoter comprises the polynucleotide in technical scheme 58.

60. An expression vector comprising the DNA construct in technical scheme 59.

61. A plant containing at least a DNA construct consisting of a heterologous target nucleotide sequence linked to a promoter, wherein said promoter comprises SEQ ID NO: 25 or 75.

62. The plant of claim 61, wherein said DNA construct is stably integrated into the plant genome.

63. The plant according to claim 61 or 62, wherein the plant is a dicotyledonous plant.

64. The plant according to claim 61 or 62, wherein the plant is a monocot.

65. The plant according to technical solution 63, wherein the monocot is corn, wheat, rice, barley, sorghum or rye.

66. The plant of technical solution 61 or 62, wherein the target DNA sequence encodes a polypeptide.

67. A method for regulating the expression of a target nucleotide sequence, said method comprising introducing into a plant a DNA construct containing a target heterologous nucleotide sequence linked to a promoter consisting of the nucleotide sequence in technical scheme 58 .

68. The method of claim 67, wherein said DNA construct is stably integrated into the plant genome.

69. The method according to technical scheme 67 or 68, wherein the plant is a dicotyledonous plant.

70. The method according to technical solution 67 or 68, wherein the plant is a monocot.

71. The method of technical solution 70, wherein the monocotyledonous plant is corn, wheat, rice, barley, sorghum or rye.

72. The method in technical scheme 67 or 68, wherein the target DNA sequence encodes a polypeptide.

The following examples are illustrative and not limiting.

Example

Example 1. Cloning and gene characterization of ZmlPT1

In the following we describe the identification and characterization of an IPT polypeptide from maize named ZmlPT1.

Materials and methods: The Mo17BAC library was screened with the 3' end fragment of ZmlPT1 cDNA from B73, identified by sequence identity to Agrobacterium ipt. One of the positive clones was digested with HindIII and subcloned into pBluescript. Recombinant plasmids were screened by clone screening using the 3' terminal fragment as a probe. One positive clone was sequenced. Samples for RT-PCR were harvested from 3 independent plants. RT-PCR was performed with 5 μg of total RNA by Invitrogen's ThermoScript RT-PCR system. The reverse transcription mix was subjected to PCR with primers designed to span intron-exon-intron junctions to avoid amplification of genomic DNA.

result:

A. Deduced protein sequence:

Two putative maize ipt ESTs were identified with deduced amino acid sequence similarities to Agrobacterium (data not shown), Arabidopsis and petunia IPT proteins (Fig. 1). The full-insertion sequencing results of these two ESTs coded that they were identical, and the corresponding cDNA was named ZmIPT1 (SEQ ID NO: 22).

Figure 1 provides an amino acid alignment of ZmlPT1, Arabidopsis and petunia cytokinin synthases. Asterisks indicate amino acids conserved in multiple cytokinin synthases. Underlined amino acids indicate the putative ATP/GTP binding site (at amino acids 84-90). As shown in Figure 1, the putative ZmlPT1 protein sequence contains the exact conserved sequence isolated from the Arabidopsis gene used by Takei et al. (2001) J. Biol. Chem. 276: 26405-26410

GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST] (SEQ ID NO: 32) (wherein x represents any amino acid residue and [ ] represents Any amino acid shown in [], x{m,n}m to n amino acids). Note that ZmlPT1 also has a putative ATP/GTP binding site at amino acids 51-58. In addition, the length of ZmlPT1 is very similar to that of AtIPT4 and Sho genes. In addition, a specific zinc finger-like element (CxxCx{12,18}HxxxxxH) (SEQ ID NO: 33) found in all eukaryotic tRNAIPTs, which is required for binding tRNA molecules, is absent in ZmIPT1.

The ZmlPT1 sequence shares 21.9% amino acid sequence identity (34.1% similarity) with full-length Sho (cytokinin synthesis protein in petunia); 10.8% identity (21.2% similarity) with full-length ipt (Agrobacterium ); with full-length AtIPT1 (Arabidopsis thaliana) with 24.7% identity (34.8% similarity); with full-length AtIPT2 (Arabidopsis thaliana) with 35.6% identity (45.3% similarity); with full-length AtIPT3 (Arabidopsis thaliana) with 22.4% identity (34.6% similarity); with full-length AtIPT4 (Arabidopsis thaliana) with 20.7% identity (31.6% similarity); with full-length AtIPT5 (Arabidopsis thaliana) with 22.7% identity (35.7% similarity); 21.8% identity (36.4% similarity) with full-length AtIPT6 (Arabidopsis); 23.4% identity (33.1% similarity) with full-length AtIPT7 (Arabidopsis); Full-length AtIPT8 (Arabidopsis thaliana) has 26.3% identity (35.9% similarity) and 18.9% identity (31.2% similarity) to full-length AtIPT9 (Arabidopsis thaliana).

ZmlPT1 sequence variants are provided. SEQ ID NOS: 22, 23 and 24 correspond to the nucleotide and amino acid sequences of ZmlPT1 derived from the cut sequence of the Mo17 genomic clone. SEQ ID NOS: 26, 27 and 28 are variants of the ZmlPT1 sequence from the full-length EST sequencing of B73. An alignment of ZmlPT1 and its variants is shown in FIG. 3 . These sequences share 98% amino acid sequence identity.

B. Gene structure

The Mo17 BAC library was screened by probes corresponding to the two ESTs, and 4 identical clones were screened. The 11 kb HindIII fragment of one of the clones was subcloned into pBluescript and sequenced. Alignment of the entire insert sequence of EST clones showed that there were 6 introns. Interestingly, both the AtIPT4 gene from Arabidopsis and the Sho gene from Petunia do not contain introns. The gene sequence of ZmlPT1 is listed as SEQ ID NO: 21.

Example 2. Gene expression of ZmlPT1

One of the identified ZmlPT1 ESTs was from the B73 embryo library and the others were from the developing root library. To obtain the expression levels of ZmlPT1, a Lynx database search was performed. An ideal tag was found at 231 bp from the poly A tail at the 3' end of the gene. Tissue type, number of library hits and average ppm are listed in Table 1. Expression was lower in most organs but higher in seedling and embryo libraries. In the embryonic library, expression of DAP was higher at 10DAP than later developmental stages (Fig. 4).

Table 1 The number and average ppm value of Lynx library containing ZmlPT1 tag.

organization type #Lynx library Average ppm Seedlings 1 29 ear 5 4 Endosperm 2 8 Embryo 4 10.75 stem 1 9 leaf 2 3 root 2 2.5

The expression profiles of ZmIPT in various maize organs during grain development were detected by RT-PCR. No amplification product was detected after 20 cycles, indicating that the expression of the gene was very low. However, after 30 cycles, a band of appropriate size could be amplified (Figure 5). Figure 5A shows that ZmlPTl transcripts are present in the ovaries of mature plants and in leaves, mesocotyls and roots of seedlings. This distribution suggests a preference for expression of this gene in meristem-like and rapidly developing tissues. In developing B73 grains (Fig. 5B), ZmlPTl transcripts were strongly present from 0 to 10 DAP and then decreased at 15 DAP. The expression profile of ZmlPT1 correlates with the known profile of cytokinins in developing grains, peaking during the lag phase. Accumulation of cytokinins initiates early cell division in endosperm and embryo development.

Likewise, ZmlPT1 labeling was only detected in the cell division zone of leaves and in BA-treated leaves. The distribution of this transcript indicates a partial tropism for ZmlPT1 expression in meristem-like and rapidly developing tissues, suggesting that maize roots and developing grains are the primary sites of cytokinin synthesis.

Example 3. ZmIPT2, ZmIPT4, ZmIPT5, ZmIPT6, ZmIPT7, Isolation and Characterization of ZmlPT8 and ZmlPT9

Six possible reading frames generated from the AtIPT1 and AtIPT3 to AtIPT8 protein sequences against the maize genome sequence were blasted, searching for a degree of similarity. Since the rice and maize genomes have a large degree of synteny, the same method was used to optimize this search against the rice genome database. The GSS maize database was then searched again with rice sequences with an E-value of at least 200. Because the GSS database was not arranged into contigs, the sequences obtained with an E-value of at least 150 were merged and aligned by Sequencher.

Eight contigs (ZmlPT2 to ZmlPT9) encoding putative CK synthetases could be identified by this method, six of which had open reading frames without introns. The translated proteins corresponding to these putative genes contain 320 to 380 amino acids, which is consistent with the expected size of plant IPT proteins. The alignment of the corresponding proteins is shown in Figure 1. The deduced protein sequences of the new ZmlPT genes (except ZmlPT8) contain the exact sequences found in different species of IPT proteins. The sequence GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST] (wherein x represents any amino acid residue, and [] represents the Any amino acid, x{m,n}m to n amino acids) (SEQ ID NO: 32) was also found in ZmIPT1, which was previously used by Kakimoto and Takei to isolate the IPT gene of Arabidopsis thaliana. The homology with other plant IPT proteins is 40%.

The top BLAST matches to the full-length amino acid sequence identities and similarities of ZmlPT2, ZmlPT4, ZmlPT5, ZmlPT6, ZmlPT7, ZmlPT8, and ZmlPT9 are listed in Table 2 below.

Table 2

Maize IPT sequence for ZmlPT2 at amino acids 17-24, for ZmlPT4 at amino acids 72-79, for ZmlPT5 at amino acids 57-64, for ZmlPT6 at amino acids 55-62, for ZmlPT7 at amino acids 23-30, for ZmlPT8 at amino acids 83-90 Also has a putative ATP/GTP binding site.

The polypeptides encoded by the ZmlPT polynucleotides have sequence similarity to known proteins. For example, the polypeptide encoded by nucleotides 821 to 3 of ZmlPT9 has 55% amino acid sequence identity to amino acids 48 to 327 of tRNA isopentenyltransferase from Arabidopsis (GenBank Accession No. BAB59048.1). The polypeptide encoded by nucleotides 821 to 3 of ZmlPT9 has 55% amino acid sequence identity to amino acids 48 to 327 of the putative IPP transferase from Arabidopsis (GenBank Accession No. AAK64114.1). The polypeptide encoded by nucleotides 821 to 3 of ZmlPT9 has 55% amino acid sequence identity to the analogous amino acid 48 to 327 of the Arabidopsis IPP transferase (GenBank accession number AAM63091.1). The polypeptide encoded by nucleotides 839 to 3 of ZmlPT9 has 36% amino acid sequence identity to amino acids 28 to 278 of the putative tRNA sigma-2-isopentenyl pyrophosphotransferase from Arabidopsis thaliana (GenBank Accession No. YP_008242.1). The polypeptide encoded by nucleotides 824 to 3 of ZmlPT9 has 35% amino acid sequence identity to amino acids 17 to 248 of tRNA isopentenyl pyrophosphotransferase of S. pneumoniae R6 (GenBank Accession No. NP_358182.1). The polypeptide encoded by nucleotides 818 to 3 of ZmlPT9 has 34% amino acid sequence identity to amino acids 2 to 231 of tRNA σ(2)-isopentenyl pyrophosphotransferase (GenBank accession number Q8CWS7). The polypeptide encoded by nucleotides 818 to 3 of ZmlPT9 has 34% amino acid sequence identity to amino acids 2 to 231 of tRNA isopentenyl pyrophosphotransferase of S. pneumoniae R6 (GenBank Accession No. NP_345176.1). The polypeptide encoded by nucleotides 818 to 3 of ZmlPT9 has 34% amino acid sequence identity with amino acids 31 to 275 of the tRNA σ(2)-isopentenyl pyrophosphotransferase of Chlamydia guinea pigs (GenBank accession number AAP05599.1) . The polypeptide encoded by nucleotides 818 to 435 of ZmlPT9 has 48% amino acid sequence identity with amino acids 6 to 133 of tRNAσ(2)-isopentenyl pyrophosphotransferase of Xylella fastidiosa 9a5c (GenBank accession number NP_297383.1 ). The polypeptide encoded by nucleotides 818 to 435 of ZmlPT9 has 48% amino acid sequence identity to amino acids 6 to 133 of tRNA σ(2)-isopentenyl pyrophosphotransferase of Xylella fastidiosa Dixon (GenBank accession no. Bacillus Dixon).

Example 4. Isolation of ZmlPT2 and ZmlPT2 Genes from Mo17 and B73 Maize Lines Molecular identification of

Materials and methods:

Plant material: Maize (Zea mays) variants B73 and Mo17 were used in this study. Samples of wild plants were harvested at different stages of development and stored at -80°C. Grain samples were harvested every 5 days from 0 to 25 DAP and separated by cutting either whole grain (0DAP), pedicel, nucellus and pericarp (5DAP), pedicel, nucellus, endosperm/embryo sac and pericarp (10DAP) or pedicel , embryo, endosperm and pericarp (15, 20 and 25DAP). Tissue corresponding to 2 to 4 different ears was collected. Cells in maize were studied using sample series harvested every DAP from 0 to 5 (whole kernel), from 6 to 15 and 20, 27 and 34 DAP (seed without husk) or 20, 25, 30 and 35 DAP (pedicel) Expression profile of the cleavin oxidase 1 gene (Ckx1) (Brugière et al., 2003, supra).

Transformation studies with Columbia wild-type Arabidopsis.

PCR: ZmlPT2 coding sequence was PCR amplified from B73 and Mo17 genomic DNA. Primers ZmlPT2-5'(5'-ATCATCAAGACA ATGGAGCACGGTG -3') (SEQ ID NO: 78) and ZmlPT2-3'(5'-CGTCCGCTAGCTACTTA TGCATCAG -3') (SEQ ID NO: 79) were designed based on the GSS contig sequence coding sequences are underlined). As part of the pathway cloning procedure, primers ZmlPT2-5-Gateway (5'-GGGGACAAGTTTGTACAAAAAGCAGGCTCAATGG-AGCACGGTGCCGTCGCCG-3') (SEQ ID NO: 80) and ZmlPT2-3-Gateway (5'-GGGGACCACTTTGTACAA-GAAAGCTGGGTCTTATGCATCAGCCACGGCGGTG-3') ( SEQ ID NO:81) Amplifies the att-flanked ZmlPT2 fragment.

In each case, step-down PCR (GeneAmp PCRSystem 9700) was performed with the following cycling conditions: 94°C 2min (1 cycle), 94°C 30s, 65°C 45s and 72°C 1min 30s (5 cycles, each cycle annealing temperature Decrease 1°C), 94°C for 30s, 60°C for 45s and 72°C for 1min 30s (30 cycles), 72°C for 7min, and stop at 4°C. Pfu Ultra Hotstart DNA polymerase (Stratagene) with a low average error rate (less than 0.5% per 500-bp amplified fragment) was used.

The PCR product was loaded on an agarose gel containing ethidium bromide (1:10000, v/v). Use QIAquick PCR purification kit (QIAgen) to recover the band corresponding to ZmlPT2 gene att-side ZmlPT2 gene.

DNA and RNA extraction: Genomic DNA was extracted from B73 and Mo17 plant samples at V3-4 stages according to Dellaporta et al. (1983) Plant Mol Biol 1:19-21 and stored at -20°C. Total RNA was prepared according to Verwoerd et al. (1989) Nucleic Acid Res 17:2362 using a hot phenol extraction procedure and stored at -80°C. Grain development sequence samples were purified with RNeasy Mini Protocol for RNA Cleanup (QIAgen) and eluted with 50 μl DEPC water. Purity of RNA preparations was assessed using optical density (DO) at 260 and 280 nm, and RNA and DNA concentrations were determined.

Southern blots, Northern blots and hybridization: For Southern blots, run digested genomic or BAC clone DNA on a 0.8% agarose gel at 110V, migrate in a 1:10000 (v/v) ethidium bromide solution - Staining was performed in TAE buffer and then transferred as follows. For Northern blots, ethidium bromide was added to denatured RNA samples and electrophoresed at 80V on a 1.5% denaturing agarose gel (Brugière et al. (2003) Plant Physiol. 132:1228-1240). Blotting was performed with a Turbo-blotter (Schleicher & Schuell) according to the instruction manual. After transfer, nylon membranes (Nytran plus, Schleicher & Schuell) were crosslinked with Stratalinker (Stratagene) and baked at 80°C for 30min. Primers were labeled with [α- ^32P ]-dCTP by random extension (Rediprime IIRandomPrime Labeling System, Amersham Biosciences) and purified with QuickSpin Columns (Roche). Hybridize with ExpressHyb hybridization solution (BD Biosciences) at 65°C for 16h, and wash the membrane under the stringent conditions as described above (0.1xSSC, 0.1% SDS) (Brugière et al. (2003) Plant Physiol.132: 1228-1240 ). Relative transcript abundance was quantified by imaging software (ImageQuant, Molecular Dynamics) with a phosphor imager (MD860, Molecular Dynamic).

BAC subcloning: BAC clones were digested and subcloned into pBluescript SK+. This plasmid includes a multiple cloning site between the lacZ gene and its promoter. The lacZ gene is usually used as a reporter gene because it encodes galactosidase, which can be hydrolyzed by X-gal enzyme to produce a black-blue precipitate. Plasmids containing the BAC fragment inserted at the multiple cloning site rendered the bacteria white due to the inability to synthesize this enzyme. Clones containing BAC subclones can thus be screened for further screening by PCR or Southern blotting.

result:

Isolation of the ZmlPT2 gene from maize genomic DNA: Genomic DNA from two different maize varieties, B73 and Mo17, was extracted and the ZmlPT2 coding sequence was amplified by descending PCR. ZmlPT2 gene amplification products were used as probes for Southern and Northern blotting experiments. The ZmlPT2 CDS was cloned into pDONR221 (Invitrogen) and sequenced. The Mo17 sequence is 100% homologous to the GSS contig sequence. The homology between the B73 and Mo17 genes at the nucleotide level was 98.8%. Differences between the two genes at the nucleotide level resulted in 3 amino acid changes at the protein level (96.1% identity). The nucleotide and amino acid sequences of ZmlPT2 are SEQ ID NO: 3 and 2, and the nucleotide and amino acid sequences of ZmlPT2 variants are SEQ ID NO: 76 and 77. The comparison of ZmlPT2 and ZmlPT2 variants showed that the differences of the polypeptides occurred at amino acid 125 (A→L), amino acid 138 (Q→R) and amino acid 193R→H.

Mapping of the gene in the maize genome: To determine whether ZmlPT2 is a single-copy or multi-copy gene in maize, B73 and Mo17 genomic DNA was digested with HindIII, EcoRI, and EcoRV and electrophoresed in a gel as described in the Materials and methods section Perform blotting. The membrane was probed with a previously extracted ZmlPT2 genomic fragment. An autoradiograph of the film is shown in FIG. 6 .

Each individual band of this digestion suggests that ZmlPT2 is more likely a single-copy gene. For the HindIII fragment identified by BAC cloning. To obtain additional information on the physical location of the ZmlPT2 gene in the maize genome, the oat-maize chromosomal addition line was used (Ananiev et al. (1997) Proc. Natl. Acad. Sci. 94:3524-3529). PCR was performed with ZmlPT2-5' and ZmlPT2-3' primers as described in the Materials and Methods section (data not shown). The expected size of the amplified fragment is 995 bp. The B73 genomic DNA sample was used as a positive control, while the oat genomic DNA and water were used as negative controls.

The data indicated that amplification of the ZmlPT2 gene was seen only in chromosome 2 oat-maize addition lines. This result was confirmed by bioinformatics methods, and the location on chromosome 2 was determined. The ZmlPT2 sequence was first used to screen B73 and Mo17 public and personal bacterial artificial chromosome (BAC) libraries. Positive clones were identified and BAC contigs identified by FPC Contig Viewer. Through this strategy, markers including several MZA markers physically mapped on maize chromosome 2, bin 4 (data not shown), were identified which allowed the experimental map of the ZmlPT2 gene to be determined by the OMA line.

Example 5. ZmlPT2, ZmlPT4, ZmlPT5, ZmlPT6 and ZmlPT8 gene expression

To obtain the expression levels of the various ZmlPT sequences, the Lynx database was searched. Tissue types, number of library matches and average ppm are listed in Tables 3-7.

As shown in Table 3, expression of ZmlPT2 is restricted to grain tissue and is associated with initiation of cytokinin synthesis in grain as described by Brugière et al. (2003) Plant Physiol. 132(3):1228-1240. Figure 9 provides a graphical representation of the ppm values of ZmlPT2 in the Lynx embryo library. The lower expression of other ZmlPT genes is consistent with a possible function of the cytokinin synthase in other tissues such as root, meristem and endosperm. See Tables 4-7 below.

Table 3. Lynx libraries with ZmlPT2 tag and ppm values.

organization type #Lynx library average ppm Endosperm＜10DAP 2 393 Corn endosperm＞10DAP 1 5 Embryo 2 17 Whole grain <10DAP 2 10 Whole grain > 10DAP 4 11 ear 2 20 Peel 1 5 pith 1 5

Table 4. Lynx library containing ZmlPT4 and ppm values.

Table 5. Lynx libraries with ZmlPT5 tag and ppm values.

Table 6. Lynx libraries with ZmlPT6 tag and ppm values.

Table 7. Lynx library with ZmlPT8 tag and ppm values.

Example 6. Expression of ZmIPT2 Gene in Maize Tissue

The expression profile of ZmlPT2 in different organs at different stages was studied to provide information on its role in cytokinin synthesis. Based on Lynx data (as described in Example 5), expression of ZmlPT2 appears to be restricted to developing grains (Figure 9). To obtain the overall information of ZmlPT2 expression in maize and validate the Lynx data, RNA was extracted from different B73 tissues—leaves, stems, roots, and whole grains—at 0, 5, 10, 15, 20, and 25 DAP. 40 μg of total RNA from each sample was stained with ethidium bromide and loaded onto an agarose gel. The obtained band was hybridized with [a- ³² P]-dCTP-labeled ZmlPT2 probe. In the second hybridization, a cyclophilin probe was used as a loading control. After quantification with a phosphor-imager, the expression ratio of ZmIPT2 was calculated after comparison with cyclophilin. Cyclophilins are thought to be constitutively expressed in different organs (Marivet et al. (1995) Mol Genet Gen 247:222-228. The results are shown in Figure 7. The levels of ZmlPT2 transcripts detected in leaves, stems and roots were very high. low, but higher levels in the grain, with lower expression at 0 DAP, increased expression from 5 to 10 DAP, and decreased expression from 15 to 25 DAP. This expression profile not only validates the Lynx data, but correlates with the appearance of CK in the grain Associated with disappearance (see Example 5).

To obtain a precise expression profile of ZmlPT2 in grains, the levels of ZmlPT2 transcripts were determined in 0- to 5-DAP husk-bearing grains, 6- to 34-DAP huskless grains, and 6- to 42-DAP peduncle alone. See Figure 8. Gels were loaded with 40 μg of total RNA, stained with ethidium bromide, and blotted on nylon membranes as previously described. The membrane was hybridized with a 32P-dCTP-labeled ZmlPT2 probe. The results of the Northern experiment with the "seed without peel" samples showed that the expression level of ZmIPT2 was very low at 0 and 4DAP. Expression increases from 5 to 8 DAP, peaks at 8 DAP, then decreases until 14 DAP. At later stages (15 to 34 DAP), transcript levels appear to start increasing again. But it is not clear whether this is the result of reduced cyclophilin expression. This phenomenon was also found in Ckx1 expression (Brugière et al. (2003) Plant Physiol 132: 1228-1240). In Northern experiments with pedicel samples, transcript levels increased sharply from 6 to 10 DAP, peaked at 10 DAP, and then slowly decreased until 15 DAP. Again in this experiment transcript levels appeared to increase again at a later stage, but it is not clear whether this is due to decreased cyclophilin expression. In this experiment, the sample corresponding to "seeds without pedicels at 9DAP" was used as a control for comparison with the other blots. The relative expression at 9DAP was 4-fold that of the control, indicating that the expression of ZmlPT2 was higher in the pedicel than in the rest of the seed. This difference is consistent with the fact that CK levels are 2-fold higher in the pedicels than in the rest of the seeds (Brugière et al. (2003) Plant Physiol 132: 1228-1240).

Relative transcript levels were compared to ZR concentrations determined in the same samples (solid line in Figure 8). Interestingly, ZmlPT2 expression overlapped well with ZR accumulation in the pedicel, whereas it slightly preceded the ZR peak in other parts of the seed, including later stages.

Taken together, these results suggest that ZmlPT2 is transiently expressed in both the pedicel and the rest of the seed during grain development, and that its expression profile parallels ZR levels in the grain is consistent with a role for ZmlPT2 as a CK synthase gene.

The expression of ZmlPT2 in different grain tissues was also investigated. In this study, segmented grain samples ranging from 0 to 25 DAP were used. Samples were collected from fields in Johnston, Iowa, USA. The grain is divided according to stage into different parts (pedicel, nucellus, endosperm/embryo sac, endosperm, embryo and pericarp). Gels were loaded with 30 μg of purified total RNA, stained with ethidium bromide, and blotted on nylon membranes.

The results shown in Figure 11 indicate that ZmlPT2 transcript levels are more abundant in the pedicels than the rest of the seeds. Especially at 15, 20 and 25 DAP, some expression was also found in embryo samples at this time. But at 10 DAP, ZmlPT2 transcripts were present in the same amount in the developing endosperm/embryo sac and pedicel. Combining the facts that 1) cytokinins are more abundant in the pedicel than the rest of the seed and 2) cytokinin oxidase transcripts and activity are also more abundant in this organ than the rest of the seed (Brugière et al. (2003) Plant Physiol. 132:1228-1240), these results indicated that the pedicel is more likely to be the main site of CK synthesis. Recent data on IPT expression in Arabidopsis led us to speculate that expression of ZmlPT2 may occur in phloem cells responsible for CK synthesis, localized to vascular bundles that transport towards developing grains. ZmlPT2 transcripts were found in both the developing endosperm and embryo when cell division in these tissues was most active. This again supports a role for ZmlPT2 as a CK synthesis protein that catalyzes CK formation in rapidly dividing/developing tissues such as endosperm and growing embryos of 10DAP, and that drives pool strength in pedicels to support grain growth.

Example 7. Expression and purification of ZmIPT2 polypeptide derived from Escherichia coli

Materials and methods:

Recombinant protein purification, gel electrophoresis and Western blotting: Escherichia coli BL21-AI (Invitrogen) containing the pDEST17-ZmIPT2(Mo17) plasmid was cultured overnight. Fresh LB medium was inoculated with a 1/200 dilution of the culture, and the bacteria were cultured at 37°C for 2h, then induced with 0.2% L-arabinose, and cultured for 2 to 4h. Bacterial protein extracts were prepared as described by the supplier and electrophoresed in 12.5% polyacrylamide gels under denaturing conditions. His-tagged proteins were purified from crude protein extracts using Ni-NTA agarose solution according to the supplier's recommendation (Qiagen). After electrophoresis, proteins were either gel-stained by GelCode (Pierce) or blotted on polyvinylidene fluoride (PVDF) membranes using an electroporation procedure. Western blotting was performed with mouse anti-histidine monoclonal antibody (Sigma-Aldrich) and sheep anti-mouse IgG antibody coupled to alkaline phosphatase as previously described (Brugière et al. (1999) Plant Cell 11: 1995-2012 ).

result:

Cloning of the ZmlPT2 coding sequence in a Gateway system compatible vector: To study the function of the ZmlPT2 protein in vitro and in vivo two methods were used. The first was to express and purify the tagged ZmlPT2 protein in E. coli, and the second was to transform Arabidopsis calli with a construct promoting the overexpression of the ZmlPT2 gene under the control of the cauliflower chimeric virus 35S promoter. The Gateway system of molecular cloning was used for this purpose.

Gateway technology was used to construct protein expression vectors (E coli Expression System with Gateway Technology kit, Invitrogen) and Arabidopsis transformation vectors (MultisiteGateway Three-Fragment Vector Construction Kit, Invitrogen). The first step is to add a specific att sequence to the previously extracted ZmlPT2 fragment. This can be achieved by PCR amplifying this fragment with a pair of primers with appropriate att sites specially designed to flank the gene.

After these specific sites were added to the flanks, the new gel-extracted fragments were inserted into the donor vector (pDONR221) by recombination. Recombination is catalyzed in vitro by BP clonase. The resulting vectors were checked by digestion with multiple restriction enzymes and migration in agarose gel electrophoresis. Digested fragment sizes corresponded to the expected lengths of digested products for each enzyme. The Mo17 and B73 ZmlPT2 genes were cloned into their respective donor vectors and the inserts were sequenced using M13 forward and reverse primers. The Mo17 clone is 100% homologous to the GSS contig sequence and thus can be used to construct expression and transformation constructs.

In vitro study: Expression of ZmlPT2 protein in Escherichia coli: The tagged ZmlPT2 protein was expressed in Escherichia coli. In addition to the transcriptional activation of the ZmlPT2 gene in E. coli through T7 promoter induction, this method also added a 6-histidine tag to the N-terminus of the recombinant ZmlPT2 protein, which can be used for its purification. The expression vector was generated by recombination of the ZmlPT2 coding sequence between pDONR221-ZmlPT2 and pDEST17 (Invitrogen).

The transcriptional fusion of 6xHis-tag and ZmlPT2 was sequenced, and Escherichia coli BL21-AI strain was transformed with the expression vector. These cells contain an expression system regulated by L-arabinose that induces the expression of T7 RNA polymerase. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) was also used to detect protein extracts collected at different times (2h and 4h) after T7RNA polymerase induction. Uninduced samples were collected as a negative control together with the expression of GUS protein with and without induction. The gel was stained with Coomassie blue, showing the presence of the protein.

After electrophoresis, induced and uninduced samples were blotted on PVDF membranes. Western blotting was performed to confirm that the induced protein contained a His-tag. For this purpose, we used murine antibodies against polyhistidine peptides. These antibodies only recognize the tagged protein, which is then recognized by an anti-mouse IgG antibody with alkaline phosphatase. The presence of recombinant protein can thus be identified by a reaction that converts the colorless substrate to a purple product that precipitates on the membrane.

Two bands can be observed, one of the expected size (about 37 kDa) and the other slightly larger (about 40 kDa), both of which are induced by L-arabinose. These two bands may be due to the addition of additional basic residues, which increase the positive charge of the protein, resulting in a change in mobility. It may also be due to the presence of covalently bound cofactors in the E. coli expressed protein. To account for these two possibilities, tryptic digests of each purified band were analyzed by mass spectrometry. In order to further identify these two bands, the protein needs to be further purified.

Purification of His-tagged ZmlPT2 protein: The presence of 6xHis tag allows affinity purification of this protein by Ni-NTA resin column. The crude extract was loaded onto the column and the flow through was collected. After washing the column several times, the protein is eluted with a solution containing imidazole, which has a high affinity for the column and thus releases the protein from the column. Samples were collected at each step of the purification and subjected to gel electrophoresis and staining as described above.

Experiments showed that the protein was expressed in soluble form, as it was present in both the supernatant and the effluent, but very little in the pellet. Protein is in the second and third elution fractions. The amount of ZmlPT2 protein in the fractions was estimated to be 70 to 80% of the total protein relative to the volume of the second solution.

Western blots were performed with the same samples. The results showed that the protein was present in very small amounts in the pellet, mostly in solution (flow-through). A strong signal in the effluent indicates that the amount of ZmlPT2 protein in the crude extract exceeds the capacity of the column.

In addition, the ZmlPT2 protein was expressed with a C-terminal tag, allowing ZmlPT2 to be purified as a single band on SDS-PAGE. The purity of the fragments is close to 100%. Furthermore, the fraction was found to have DMAPP:ADP and DMAPP:ATP prenyltransferase activity.

Example 8. In vitro study of overexpression of ZmIPT2 gene in Arabidopsis

Materials and methods:

In vitro culture : Different media were used for Arabidopsis germination, callus culture and regeneration (Kakimoto (1998) J. Plant Res. 111:261-265). The media used are as follows:

■5000X CIM (callus induction medium) hormone mixture: 2.5mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D), 0.25mg/ml kinetin and 5mg/ml biotin dissolved in dimethyl in sulfoxide (DMSO).

■ 500X vitamin compound: 50mg/ml inositol. 10mg/ml Thiamine-HCl, 0.5mg/ml Vitamin B6-HCl and 0.5mg/ml Niacin.

GM (germination medium): 1L containing 4.3g Murashige and Skoog's medium salt components (Sigma), 10g sucrose, 2ml 500X vitamin mixture, 10ml 5% 2-(N-morpholine)-isethionic acid (MES , adjusted to pH 5.7 with KOH) and a mixture of 3 g Phytagel (Sigma), autoclaved.

CIM (Callus Induction Medium): 1L mixture containing 3.08g Gamborg's B5 medium salt component (Sigma), 20g glucose, 2ml 500X vitamin mixture, 10ml 5% MES (adjusted to pH 5.7 with KOH) and 3g Phytagel, Autoclave and add 200 μl of 5000X CIM Hormone Mix.

■AIM (Agrobacterium Infection Medium): prepared by omitting Phytage from CIM.

■ WASHM (washing medium): Phytagel was omitted from GM, and 100 mg/l cefotaxime sodium was added.

Selection medium for transformed callus:

■GM+IBA (GIBA): GM plus 100mg/l cefotaxime, 50mg/l carbenicillin, 3mg/l bialaphos and 0.3mg/l indolebutyric acid (IBA).

GM+IBA+Z (GIBAZ): GM plus 100mg/l cefotaxime, 50mg/l carbenicillin, 3mg/l bialaphos, 0.3mg/l indolebutyric acid (IBA) and 1mg/l trans Formula zeatin (tZ).

The purpose of the experiment is to test the effects of auxin and cytokinin on root and shoot regeneration, prepare GM, and add different amounts of hormones. Prepare 25 media containing different concentrations of tZ and IBA, each hormone set at 0, 100, 300, 1000 and 3000 ng/ml.

Sterilized Arabidopsis seeds were germinated in GM medium and grown at 23°C under continuous light. For the above experiments, hypocotyls of 15-day-old seedlings were cut with a scalpel and cultured in 25 medium at 23°C under continuous light for 3 weeks. For experiments using callus, hypocotyls were grown in CIM for 10 to 12 days under the same conditions.

Arabidopsis callus transformation: Soak the induced callus in AIM Agrobacterium suspension (0.2 OD ₆₀₀ ) for 5 minutes. Most of the liquid was removed on filter paper, and the callus was placed in CIM medium and cultured at 23°C under continuous light for 2 days. Then the calli were rinsed well in WASHM medium and cultured on GIBA or GIBAZ medium for about 3 weeks.

Cloning: For the sustained expression of ZmlPT2 in Arabidopsis using the Gateway system, a construct containing the gene under the control of the cauliflower chimeric virus 35S promoter was constructed. A pathway clone containing the 35S promoter was constructed with the pDONR-P4-P1R plasmid. When these 3 elements are obtained, multisite recombination is performed with 3 donor vectors and a fourth vector called the destination vector.

LR cloning enzyme can perform orderly "3-site" recombination between the plasmid carrying the promoter, the target gene and the terminator, and the binary vector containing the left and right border regions of the Ti plasmid and the BAR drug resistance gene. The resulting constructs were verified by digestion with restriction enzymes, gel electrophoresis, and comparison of the size of the digested fragments to the expected digest product.

The final construct contained the 35S-ZmIPT2-PINII sequence and included the BAR gene. This gene acts as a selectable marker, conferring herbicide resistance to transformed plant cells.

Transformation of Agrobacterium: The next step is to transform the plasmid containing the 35S-ZmIPT2-PINII construct into Agrobacterium tumefaciens (LBA4044). This plasmid contains the genes (vir genes) required for infection and delivery of T-DNA into Agrobacterium tumefaciens. The two plasmids were able to recombine at their respective COS sites following bacterial electroporation (Suzuki (1999) Plant Cell Physiol 39: 1258-1268). The result of the recombination is a 48kb plasmid called a "co-integrate".

Agrobacteria containing the co-integrate were tested by a quality control procedure. The procedure involves extraction of co-integrate plasmids, transformation into E. coli, and verification by restriction digest. This step is necessary to screen for "false recombination" of the two plasmids at the COS site resulting in a non-functional construct.

Although multiple routes were used to transform the 35S-ZmIPT2-PINII construct into Agrobacterium, no clones containing the correct co-integrate plasmid were identified. Since the 35S promoter is leaky in Agrobacterium, expression of ZmlPT2 was assumed to be lethal to Agrobacterium. The lethality of the constructs may be due to the active degradation of essential compounds of Agrobacterium. Such metabolites may arise, for example, from the isoprenoid synthesis pathway, including possible substrates for CK synthesis, such as 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBPP).

Analysis of microbial genomes and biochemical experiments established two isoprenoid synthesis pathways, methylvalerate (MVA) and non-methylvalerate (1-deoxyxylulose-5-phosphate, DXP or 2 -C-methyl-D-erythritol-4-phosphate, MEP) pathway. The DXP pathway is present in some bacteria and plant chloroplasts. Genes encoding the non-methylvalerate (MEP) pathway are predominantly found in Gram-positive bacteria. HMBPP, a precursor of the non-methylpentanoate (MEP) pathway of isoprenoid synthesis, is a possible substrate for AtIPT7 (Takei et al. (2003) J Plant Res 116:265-9). Analysis of the gene sequence of Agrobacterium tumefaciens C58 shows that it encodes enzymes of the MEP pathway, but no enzymes of the MVA pathway (Wood et al. (2001) Science 294: 2317-2323; Goodner et al. (2001) Science 294: 232-2328 ). Based on these results, we propose that HMBPP is a substrate for the ZmlPT2 protein and that utilization of this compound by the enzyme inhibits isoprenoid formation, resulting in the inability of the bacteria to grow.

To circumvent this problem, the same construct was constructed but this time using the 35S promoter linked to the ADH1 intron to suppress expression of the ZmlPT2 gene in Agrobacterium. Agrobacteria carrying the correct co-integrates can be obtained using this construct.

result:

The root or shoot regeneration of Arabidopsis callus in the medium depends on the levels of auxin and cytokinin in the medium. Therefore, Arabidopsis hypocotyls were cultured in media containing increased levels of auxins and cytokinins. Combinations of 25 different tZ and IBA concentrations were prepared, at concentrations of 0, 100, 300, 1000 and 3000 ng/ml for each hormone, and the hypocotyls were transferred to medium as described above. After 3 weeks in the culture room, pictures of 2 representative calli were taken for each hormone combination. The results indicated that a higher auxin:cytokinin ratio favored root formation, while a higher cytokinin:auxin ratio favored shoot formation.

This experiment shows that root or shoot formation is affected by the auxin/cytokinin ratio. Auxins have a root-inducing effect, while cytokinins induce shoot formation. Based on these results, Arabidopsis calli overexpressing cytokinin synthases were unable to develop roots in auxin-only media. The function of this assay to identify putative cytokinin synthesis genes through the Arabidopsis IPT(rmr) gene was tested. Specifically, two constructs overexpressing IPT as a cytokinin synthase or GUS as a control were constructed using the pathway cloning system. Three weeks after transformation of Arabidopsis callus, root production could be seen in the calli transformed with the 35S-GUS-PINII construct, but not in the calli transformed with the 35S-IPT-PINII construct. To verify that the callus was efficiently transformed, in situ GUS staining was performed. These tissues transformed with 35S-GUS-PINII contain GUS protein and develop a blue color after incubation in a solution containing GUS substrate. These results validate the detectability of putative maize CK synthesis genes using a high-throughput assay.

Arabidopsis calli transferred to GM medium containing auxin or auxin and cytokinin for 10 days were transformed with the 35S-ADHI-ZmIPT2-PinII construct. Transformed calli were selected by adding herbicides. Obvious phenotypes developed 3 weeks after transformation. Control and 35S-ADHI-ZmIPT2-PINII calli grew identically on auxin- and cytokinin-containing media. As expected, control calli transformed with the 35S-GUS-PINII construct could regenerate roots on auxin-only media. In contrast, calli transformed with the 35S-ADH1-ZmIPT2-PINII construct failed to produce roots in this medium as did calli transformed with the 35S-IPT-PINII construct, and some calli could even regenerate branch. According to the previous preliminary experiments, these calli synthesized CK due to the expression of ZmIPT2 gene. This reduces the auxin:cytokinin ratio and inhibits root formation. These results support the conclusion that ZmlPT2 is a cytokinin synthesis gene.

Example 9. Isolation and sequencing of the ZmlPT2 promoter

To isolate the promoter of the ZmlPT2 gene, a high-throughput bacterial artificial chromosome (BAC) screening process was used. According to the sequence of ZmlPT2, 5 positive clones were isolated by PCR screening. To verify that the target gene is present on the bacterial chromosome, BAC clones were cultured and prepared. The obtained BACs were digested with HindIII and subjected to gel electrophoresis followed by Southern blotting. Blots were hybridized with [a- ^32P ]-dCTP-labeled ZmlPT2 probe. The method of Southern blotting was as described in Example 4.

Southern blots showed the presence of the ZmlPT2 sequence in all isolated BAC clones. After validation, BACs were subcloned into pBluescript digested with BamHI and HindIII. After ligation, chemically prepared competent E. coli were transformed and cultured on ampicillin LB medium. Positive clones were then screened by clonal hybridization method. Clones were transferred to nylon membranes and hybridized with the [α- ^32P ]-dCTP ZmlPT2 probe to detect clones containing the ZmlPT2 region in their plasmids. Finally, screened clones were prepared and the plasmids were sequenced with primers in the 5'OH direction. Thus, the 1354bp sequence of the upstream region of ZmlPT2 can be obtained. The 3280bp sequence of the gene promoter can be obtained by using the BAC walking strategy. The ZmlPT2 promoter sequence is SEQ ID NO:75. The ZmlPT1 promoter in SEQ ID NO: 25 was identified using the same strategy.

The promoter sequences of ZmlPT4 to ZmlPT9, and OsIPT1 to OsIPT11 can be isolated in the same manner. The sequences provided herein ZmIPT4 (SEQ ID NO: 5), ZmIPT5 (SEQ ID NO: 8), ZmIPT6 (SEQ ID NO: 11), ZmIPT7 (SEQ ID NO: 14), ZmIPT8 (SEQ ID NO: 17), and ZmIPT9 (SEQ ID NO: 20), OsIPT1 (SEQ ID NO: 47), OsIPT2 (SEQ ID NO: 44), OsIPT3 (SEQ ID NO: 62), OsIPT4 (SEQ ID NO: 64), OsIPT5 (SEQ ID NO: 50 ), OsIPT6 (SEQ ID NO: 55), OsIPT7 (SEQ ID NO: 53), OsIPT8 (SEQ ID NO: 40), OsIPT9 (SEQ ID NO: 60), OsIPT10 (SEQ ID NO: 58) and OsIPT11 (SEQ ID NO: 58) ID NO:42) includes a suitable upstream region for identification of a functional promoter sequence.

Example 10.IPT activity detection

A. Cytokinin synthesis by maize or rice IPT sequences in bacterial culture

The ability of the IPT sequences of the invention to synthesize cytokinins was determined in culture medium of bacteria known to secrete cytokinins. Enzyme activity was determined in E. coli.

Escherichia coli BL21-AI (Invitrogen) (IPT cloned in pDEST17 (Invitrogen)) containing T7 promoter:IPT sequence was cultured at 37°C for 4h, and protein accumulation was induced at 20°C for 12h in the presence of 0.2% arabinose. Microorganisms were collected by centrifugation, then buffer A (25 mM Tris-HCl, 50 mM KCI, 5 mM β-mercaptoethanol, 1 mM PMSF, and 20 μg/ml leupeptin) was added to an OD600 of 100, and Escherichia coli was disrupted by freeze-thawing. The disrupted E. coli was then centrifuged at 300,000 g for 10 minutes to recover the supernatant. 10 μl of the supernatant was mixed with buffer A containing 60 μM DMAPP, 5 μM [3H]AMP (722 GBq/mmol) and 10 MM MgCl ₂ , and incubated at 25° C. for 30 minutes. Then, 50 mM Tris-HCl (pH 9) was added to the reaction solution, and then calf intestinal alkaline phosphatase was added to a concentration of 2 units/30 μl, and incubated at 37° C. for 30 minutes for dephosphorylation reaction. The reaction solution was developed by C18 reverse thin-layer chromatography (mobile phase: 50% methanol), and the reaction product was detected by autoradiography. There is the formation of isopentenyl adenosine.

It is further known that 3H-HMBPP (4-hydroxy-3-methyl-2-(E)butenyl diphosphate) can be used as a substrate for the above detection method. See, eg, Krall et al. (2002) FEBS Letters 527:318-8, incorporated herein by reference.

B. Detection of DMAPP:ATP or ADP or AMP prenyltransferase activity

DMAPP:ATP (or ADP or AMP) prenyltransferase activity was determined by the method described by Blackwell and Horgan (1991) FEBS Lett. 16: 10-12, with some modifications. The samples tested are crude extracts and purified E. coli proteins with T7 promoter::IPT sequence. The purified protein was diluted to an appropriate concentration with dilution buffer (25 mM Tris·HCl, pH 7.5; 5 mM 2-mercaptoethanol; 0.2 mg ml ⁻¹ bovine serum albumin). The sample was mixed with an equal volume containing 25mM Tris·HCl (pH 7.5), 10mM MgCl ₂ , 5mM 2-mercaptoethanol, 60μM DMAPP and 2μM [2,8- ³ H]ATP (120GBq mmol ^-1 ), [2,8- A 2x detection mixture of ³ H]ADP (118GBq mmol ^-1 ), [2- ³ H]AMP (72GBq mmol ^-1 ) or [2,8- ³ H]adenosine (143GBq mmol ^-1 ) was mixed for isoprene Isopentenylation reaction. After incubation for a suitable time, 1/2 volume of bovine intestinal alkaline phosphatase (CIAP) mixture [0.5 Tris·HCl (pH 9.0), 10 mM MgCl ₂ and 1,000 units ml ⁻¹ CIAP (Takara Shuzo Co. Ltd., Otsu , Shiga, Japan)], the mixture was incubated at 37°C for 30min. Then 700 μl of ethyl acetate was added and the mixture was shaken. Centrifuge at 17,000xg for 2 min, recover the organic phase, and rinse with water twice. The organic phase was mixed with 10 times the volume of scintillation fluid ACSII (Amersham Pharmacia Biotech, Tokyo, Japan), and the radioactivity level was measured with a liquid scintillation counter. [ ^2,8-3H ]isopentenyladenosine (iPA) recovery was determined and used to calculate the amount of product formed. [ ^2,8-3H ]iPA was synthesized by prenylation of ATP using the purified IPT sequence, followed by CIAP treatment as described above. All determinations were repeated twice and average values were used for calculations.

To determine the K _m of ATP, the purified protein (2 ng ml ^-1 dissolved in dilution buffer) was mixed with an equal volume containing 25 mM Tris·HCl (pH 7.5), 10 mM MgCl ₂ , 5 mM 2-mercaptoethanol, 0.4 mM DMAPP and ATP (2-502 μM [2,8- ³ H]ATP, 1.22 MBqml ^-1 ) of 2x detection mix was mixed. To determine the K _m of DMAPP, the purified protein (2ng ml ^-1 ) was mixed with an equal volume of 25 mM Tris·HCl (pH 7.5), 10 mM MgCl ₂ , 5 mM 2-mercaptoethanol, 0.25-200 μM DMAPP and 200 μM [2,8- ³ ] 2x detection mix of ATP (7.07GBq mmol ^-1 ) was mixed. The mixture was incubated at 24°C for 0 min or 4 min, the reaction mixture was treated with CIAP and then extracted with ethyl acetate as described above. The value obtained at 4 min was subtracted from the value obtained at 0 min, and the difference obtained was the enzyme activity.

To confirm that the IPT sequence catalyzes the transfer of the prenyl group to ATP, ADP or AMP, the reaction products were analyzed by HPLC and mass spectrometry. Briefly, crude extracts prepared from IPTG-induced E. coli containing the pDEST17-IPT plasmid were incubated with Ni-NTA agarose beads. After the beads were thoroughly rinsed, they were resuspended in a solution containing 25 mM Tris·HCl (pH 7.5), 100 mM KCl, and 5 mM 2-mercaptoethanol. The microsphere pellet was mixed with an equal volume of 2x detection mix containing 1 mM unlabeled ATP and 1 mM DMAPP and incubated at 25°C for 1 hour with shaking. After centrifugation, the supernatant was recovered and divided into two fractions, one was treated with CIAP as described previously. Supernatants treated with or without CIAP were mixed with 3 volumes of acetone. The mixture was incubated at -80°C for 30 min, then centrifuged at 17,000 xg for 30 min to remove protein. The supernatant was dried in vacuo, and the residue was dissolved in methanol. Aliquots were separated by HPLC on a Chemcobond ODS-W column (Chemco, _Osaka , Japan) using the following program: 15 min in 20 mM _KH2PO4 , followed by 0% acetonitrile and ₂₀ mM _KH2PO4 to 80% acetonitrile and A linear gradient _of 4 mM _KH2PO4 was performed for 30 min. Fractions were collected, dried in vacuo, and the residue resuspended in ethanol. After centrifugation to remove possible salt precipitates, the solution was subjected to fast atom bombardment ionization mass spectrometry (JMS-SX102 or JEOL MStation, JEOL DATUM LTD., Tokyo, Japan).

C. Determination of shoot and root regeneration

Transformation of Arabidopsis callus was performed as follows. Transformants were selected with 3 mg/L bialaphos. Arabidopsis seeds were sterilized as described by Koncz et al. (1992) Methods in Arabidopsis Research, Sinapore, River Edge, NJ, World Scientific. Seeds were placed on GM medium and grown for 11 days at 23°C under continuous light. Hypocotyl segments were cut and cultured on CIM for 8 days. The calli were soaked in AIM Agrobacterium suspension (0.2 OD ₆₀₀ ) for 5 minutes. Most of the liquid was removed on filter paper, and the Arabidopsis was grown on CIM medium at 23°C for 2 days under continuous light. Rinse the callus well in WASHM medium, and culture it on GM+IBA or GM+Z+IBA medium for about 3 weeks. Transformants were screened with 3 mg/L bialaphos.

The medium recipes for the above transformation experiments are as follows. 5000xCIM hormone mixture contains 2.5mg/ml 2,4-D (Sigma Cat.No.D 6679); 0.25mg/ml cytokinin (kinetin) (Sigma Cat no.K 0753) and 5mg/ml dissolved in DMSO Biotin (Sigma Cat.No.B 3399. 500x Vitamin Blend with 50g/l Inositol (SigmaCat.No.I 3011); 10g/l Thiamine-HCl (Sigma Cat.No.T 3902); 0.5g/l Vitamin B6-HCl (Sigma Cat.No.P 8666) and 0.5g/l Niacin (Sigma Cat.No.N0765). GM (germination medium) (1 L) contains 4.3g MS medium salt components (SigmaCat. No.M 5524); 10g sucrose (Sigma Cat.No.S 8501); 2ml 500x vitamin mixture; 10ml 5% MES (adjusted to pH 5.7 with KOH) (Sigma Cat.No.M 2933) and 3g Phytagel (Sigma Cat .No.P 8169).Mixture autoclaved and poured on the Petri dish.CIM (callus induction medium) contains 3.08g Gamborg'sB5 medium salt composition (Sigma Cat.No.G 5768); 20g glucose ( Sigma Cat.No.G7528); 2ml 500x vitamin mixture; 10ml 5% MES (adjusted to pH 5.7 with KOH) and 3g Phytagel. The mixture was autoclaved, and after cooling, 200 μl of CIM hormone mixture was added. The mixture was then poured into Petri dishes. AIM (agrobacterium infection medium) contains CIM without Phytagel. WASHM (washing medium) is GM without Phytagel, supplemented with 100 mg/l cefotaxime sodium. GM+ (selection of transformed callus contains autoclaved GM culture medium, and then add the following components through filter paper: 1ml 100mg/ml cefotaxime (Sigma Cat.No.C 7039); 1ml 50mg/ml carbenicillin (SigmaCat.No.C 3416) and 3ml 1mg/ml dipropyl Aminophosphine. GM+IBA is to add 300 μl 1mg/ml indolebutyric acid (IBA) (Sigma Cat.No.I 7512) to the above-mentioned GM medium. GM+IBA+Z is to add 300 μl 1mg to the above-mentioned GM medium /ml IBA and 1ml 1mg/ml trans-zeatin (Z) (Sigma Cat.No.Z 2753).

In order to test the function of IPT, the IPT sequence of maize was first selected and introduced into the callus of Arabidopsis thaliana under the control of 35S promoter. Calli transformed with the control vector showed normal hormone responses. Root formation occurs only in the presence of auxin whereas shoot formation occurs in the presence of both cytokinin and auxin. In contrast, calli transformed with 35S::IPT regenerated shoots even in the absence or presence of reduced concentrations of exogenously used cytokinins. In addition, modulation of cytokinin synthesis can be assayed for changes in its various directions. Representative methods include cytokinin extraction, immunopurification, HPLC separation and ELISA quantification methods, see eg Faiss et al. (1997) Plant J. 12:401-415. See also Werner et al. (2001) PNAS 98: 10487-10492) and Dewitte et al. (1999) Plant Physiol. 119: 111-121.

D.DMAPP: Determination of tRNA Prenyl Transferase Activity

Undermodified tRNA was prepared by treating yeast tRNA (type X, Sigma-Aldrich Japan, Tokyo, Japan) with permanganate according to the method of Kline et al. (1969) Biochemistry 8: 4361-4371. 20 microliters of purified protein samples (20 ng (protein ml ⁻¹ ) in dilution buffer were mixed with an equal volume of 2X tRNA prenyltransferase assay mix (25 mM Tris-HCl, pH 7.5; 10 mM MgCl ₂ ; 5 mM 2- mercaptoethanol; 0.67 μM [1- ³ H]DMAPP, 555 GBq mmol ^-1 ; 567 A ₂₆₀ units ml ^-1 modified under-modified tRNA) at 25°C for 30 min. Add 160 μl 0.4M sodium acetate and 500 μl ethanol, in ice After standing still for 10 minutes, the tRNA precipitate was recovered by centrifugation (17,000×g for 20 minutes), rinsed with 80% ETOH, and dissolved in 30 μl sterile water. Mixed with 10 times the volume of ACSI, the radioactivity level was determined.

Example 11. Maintaining or increasing seed fixation under stress

Site-directed overexpression of the IPT sequences of the invention in developing female inflorescences increases cytokinin levels and allows developing maize seeds to achieve their full genetic potential in volume, reduce apical kernel abortion, and buffer against adverse environments Seeds are fixed. Abiotic stresses during grain development can reduce cytokinin levels. Under stress conditions, cytokinin synthesis activity may decrease, while cytokinin degradation increases (Brugiere et al. (2003) Plant Physiol. 132(3): 1228-40). Thus, in one non-limiting approach to maintaining cytokinin levels in lagging grains, linking IPT genes in 1) stress insensitivity; 2) direct expression of structural genes is predominant for developing grains; and 3) on the controllable elements that can preferentially initiate the expression of structural genes during the lagging stage of grain development. Promoters that localize expression at or near anthesis in the relevant maternal tissue may be used. Alternatively, constitutive promoters can be used.

For example, immature maize embryos from greenhouse donor plants were prepared with a gene selected from ZmIPT1-9 or OsIPT1-11, linked to the Zag2.1 promoter (Schmidt et al. (1993) Plant Cell 5:729-737), and containing Plasmid bombardment of the sequence of the selectable marker gene BAR (Wohlleben et al. (1988) Gene 70:25-37) confers on the herbicide bialaphos. Alternatively, the selectable marker gene can be on another plasmid. The conversion is performed as follows. The medium prescription is as follows.

Ears were husked and surface sterilized in 30% Clorox bleach supplemented with 0.5% micro-detergent for 20 minutes, followed by two rinses in sterile water. The immature embryos were cut, and the hypocotyls were placed under the side (on the cotyledon disc side), 25 embryos were placed on each plate, cultured on 560Y medium for 4 hours, and then aligned with the 2.5cm positioning area, ready for bombardment.

A plasmid containing the IPT sequence linked to the Zag2.1 promoter was prepared. This plasmid DNA was precipitated with _the plasmid DNA containing the BAR selectable marker on a 1.1 μm (average diameter) tungsten pellet—using the following CaCl precipitation procedure: 100 μl of water containing prepared tungsten particles; 10 μl of ( 1 μg) DNA (1 μg total DNA); 100 μl 2.5 M CaCl ₂ and 10 μl 0.1 M spermidine.

Add each reagent sequentially to the tungsten particle suspension on a multitube shaker. The final mixture was sonicated and incubated for 10 minutes with constant shaking. After precipitation, the tubes were centrifuged, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Remove the liquid again and add 105 μl of 100% ethanol to the final tungsten pellet pellet. For particle gun bombardment, tungsten/DNA particles were sonicated and 10 μl was spotted in the center of each carrier and allowed to dry for 2 minutes prior to bombardment.

Sample plates were bombarded at level #4 or #HE34-1 or #HE34-2 of the particle gun. All samples were subjected to a single bombardment at 650 PSI with a total of 10 particles/DNA prepared from each tube.

After bombardment, the embryos were cultured on 560Y medium for 2 days, and then transferred to 560R selection medium containing 3 mg/L bialaphos for subculture every 2 weeks. After about 10 weeks of selection, the selected resistant callus pellets were transferred to 288J medium to initiate plant regeneration. After somatic embryos mature (2-4 weeks), well-developed somatic embryos are transferred to germination medium and transferred to a light culture room. After about 7-10 days, transfer the developed seedlings to 272V hormone-free medium tubes and culture them for 7-10 days until the seedlings are well formed. Plants are then transferred to flat sockets containing potting soil (equivalent to 2.5" pots) and grown for 1 week in a culture room, followed by an additional 1-2 weeks in a greenhouse before being transferred to a classic 600 pot (1.6 gallon ) to maturity. Monitor the plants and score for maintaining or increasing seed fixation during the abiotic stress period. In addition, monitor the cytokinin levels of the transformants under stress (as described in Example 5c) and maintain grain growth .

Bombardment medium (560Y) containing 4.0g/l N6 basal salts (SIGMA C-1416), 1.0ml/l Eriksson's vitamin mixture (1000X SIGMA-1511), 0.5mg/l Thiamine HCl, 120.0g/l Sucrose, 1.0 mg/l 2,4-D and 2.88g/l L-proline (after adjusting the pH to 5.8 with KOH, dilute to volume with DI H ₂ O); 2.0g/l Gelrite (dilute to volume with DI H ₂ O Add) and 8.5 mg/l silver nitrate (add after the culture medium is sterilized and cooled to room temperature). Selection medium (560R) containing 4.0g/l N6 basal salts (SIGMA C-1416), 1.0ml/l Eriksson's vitamin mixture (1000X SIGMA-1511), 0.5mg/l Thiamine HCl, 30.0g/l sucrose and 2.0mg /l 2,4-D (adjust the pH to 5.8 with KOH and then use DI H ₂ O to make up); 3.0g/l Gelrite (add in after making up with DI H ₂ O) and 0.85mg/l silver nitrate and 3.0 mg/l bialaphos (both added after the culture medium was sterilized and cooled to room temperature).

Plant regeneration medium (288J) containing 4.3g/l MS salts (GIBCO 11117-074), 5.0ml/l MS vitamin stock solution (0.100g Niacin, 0.02g/l Thiamine HCL, 0.10g/l Vitamin B6 HCL and 0.40g/l glycine, with pure DI H ₂ O to volume) (Murashige and Skoog (1962) Physiol. Plant. 15: 473), 100mg/l inositol, 0.5mg/l zeatin, 60g/l sucrose and 1.0ml/l 0.1mM abscisic acid (adjust to pH 5.6 with pure DI H ₂ O to volume); 3.0g/l Gelrite (add after volume) and 1.0mg/l indole acetic acid and 3.0mg/l bis Alanphos (add after the culture medium is sterilized and cooled to 60°C). Hormone-free medium (272V) containing 4.3g/l MS salts (GIBCO 11117-074), 5.0ml/l MS vitamin stock solution (0.100g Niacin, 0.02g/l Thiamine HCL, 0.10g/l Vitamin B6 HCL and 0.40g/l glycine (diluted to volume with pure DI _H2O ), 0.1g/l inositol and 40.0g/l sucrose (diluted to volume with pure DI _H2O after adjusting to pH 5.6) and 6g/l bacteria Agar (added to volume with pure DI H ₂ O), sterilized and cooled to 60°C.

Example 12: Modulation of Root Development

For Agrobacterium-mediated transformation of maize with a plasmid designed for post-transcriptional gene silencing (PTGS) through an appropriate promoter, the method of Zhao can be used (US Patent 5,981,840 and PCT Patent Publication WO 98/32326, the contents of which are available as incorporated herein by reference). Briefly, immature embryos are isolated from maize and contacted with an Agrobacterium suspension transformable with the DNA construct. The construct comprises a CRWAQ81 root-biased promoter::ADH intron promoter connected with a hairpin structure derived from any one of the coding sequence of the ZmlPT1-9 or OsIPT1-11 polynucleotide of the present invention. Other useful constructs include hairpin constructs acting on the promoter of any of the ZmlPT1-9 or OsIPT1-11 polynucleotides of the invention. (Aufsatz et al. (2002) PNAS 99 (Suppl. 4): 16499-16506; Mette et al. (2000) EMBO J 19 (19): 5194-5201). The construct is transferred to at least one cell of at least one immature embryo (step 1: infection step). In this step, immature embryos are soaked in an Agrobacterium suspension for inoculation. Embryos are co-cultivated with Agrobacterium for a period of time (step 2: co-cultivation step); this can be done on solid medium. Co-cultivation is followed by an optional "resting" step. During the resting step, the embryos are cultured in the presence of at least one antibiotic that inhibits the growth of Agrobacterium without addition of plant transformant selection reagents (step 3: resting step). The inoculated embryos are then cultured on medium containing the selection agent; grown and the transformed callus recovered (step 4: selection step). The callus is then regenerated into plants (step 5: regeneration step).

Plants were monitored and scored for regulation of root development. Modulation of root development includes monitoring enhanced root growth of one or more root fractions, including primary roots, lateral roots, adventitious roots, and the like. Methods for determining such changes in root phylogeny are known in the art. See, eg, US Patent Application 2003/0074698 and Werner et al. (2001) PNAS 18:10487-10492, incorporated herein by reference.

Example 13. Regulation of plant senescence

ZmIPT1-9 or ZmIPT1-9 linked to a constitutive promoter, a root-biased promoter, or a senescence-promoting promoter—for example, SAG12 (Gan et al. (1995) Science 270:5244, Genbank Acc. No. U37336). DNA constructs of any one of the polynucleotides of OsIPT1-11 were introduced into maize plants as described by Zhao et al. (1998) Maize Genetics Corporation Newsletter 72:34-37, incorporated herein by reference.

For example, maize plants are obtained that contain the IPT sequence linked to the SAG12 promoter. As a control, a cytokinin-independent construct was also introduced into maize plants by the transformation method described above. The phenotype of transgenic maize plants with elevated levels of IPT polypeptides was studied. For example, plants can be monitored for increased vigor, shelf life in vases, and improved resistance to infection. The ability of plants to delay senescence can be monitored under various environmental stresses including, for example, waterlogging which often results in leaf wilt, necrosis, shedding, cessation of growth and reduced yield.

Example 14. Transformation of soybean embryos

Soybean embryos were bombarded with a plasmid containing the IPT sequence linked to the ubiquitin promoter described below. To induce somatic embryos, cotyledons 3-5 mm in length excised from immature seeds of soybean cultivar A2872 are grown on a sterilized surface at 26°C in the light or in the dark for 6 to 10 weeks in a suitable agarose medium. The somatic embryos producing secondary embryos are then excised and placed in a suitable liquid medium. After repeated screening of clusters of somatic embryos that propagated to early globular stage embryos, the suspension was maintained as follows.

In a rotary flask containing 35ml of liquid medium, at 150rpm, 26°C, under the condition of fluorescent irradiation for 16:8 hours day/night, soybean embryo stage suspension culture can be maintained. Subcultures were performed every two weeks by inoculating approximately 35 mg of tissue in liquid medium.

Soybean embryo stage suspension cultures were then transformed by particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Patent No. 4,945,050). A Du Pont Biolistie PDS1000/HE instrument (helium retrofit) was used in the transformation.

The selection marker gene that is beneficial to soybean transformation can be the transgene (Odell et al. (1985) Nature 313:810-812) containing cauliflower chimeric virus 35S promoter, the hygromycin phosphotransferase gene (from E.coli ; Gritz et al. (1983) Gene25: 179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Agrobacterium tumefaciens Ti plasmid. The expression cassette containing the IPT sequence linked to ubiquitin can be isolated as a restriction fragment. This fragment is then inserted into a specific restriction site of the vector containing the marker gene.

To 50 μl of a 60 mg/ml suspension of 1 μm gold particles was added (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1M) and 50 μl CaCl ₂ (2.5M). The pellet preparation was then shaken for 3 minutes and centrifuged for 10 seconds to remove the supernatant. The DNA-coated particles were then washed once in 400 μl of 70% ethanol and resuspended in 40 μl of absolute ethanol. Sonicate the DNA/particle suspension 3 times for 1 s each. 5 microliters of the DNA-coated gold particles were then loaded into the respective megacarrier plates.

Approximately 300-400 mg of a two-week suspension culture was placed in an empty 60 x 15 mm petri dish, and residual liquid was removed from the tissue with a pipette. Typically 5-10 plates of tissue are bombarded for each transformation experiment. The membrane rupture pressure was set at 1100 psi and the chamber was evacuated to 28 inches of mercury. Place the tissue 3.5 inches away from the protective screen and bombard 3 times. After bombardment, the tissue was split in two and returned to liquid for culture as described above.

After 5 to 7 days of bombardment, the liquid medium was replaced with fresh medium, and after 7 to 12 days of bombardment, it was replaced with fresh medium containing 50 mg/ml hygromycin. The selection medium was refreshed weekly. Seven to eight weeks after bombardment, green transformed tissue growth was observed from untransformed necrotic embryo clusters. Isolated green tissue was removed and inoculated into flasks to generate new, clonally propagated, transformed embryo suspension cultures. Each new line can be processed as an independent transformation event. These suspensions are then subcultured to maintain clusters of immature embryos or to regenerate whole plants by maturation and germination of individual somatic embryos.

Example 15. Sunflower Meristem Transformation

Sunflower meristems were transformed with an expression cassette containing an IPT sequence linked to a ubiquitin promoter as follows (see also European Patent EP 0486233, incorporated herein by reference, and Malone-Schoneberg et al. (1994) Plant Science 103: 199- 207). Ripe sunflower seeds (Helianthus annuus L.) were dehulled with a single ear thresher. Seeds were surface sterilized for 30 minutes in a 20% Clorox bleach solution with 2 drops of Tween 20 added per 50 ml of solution. Rinse the seeds twice with sterile distilled water.

Split hypocotyl explants were prepared by a modification of the procedure described by Schrammeijer et al. (Schrammeijer et al. (1990) PlantCell Rep. 9:55-60). Seeds were soaked in distilled water for 60 min after surface sterilization. The cotyledons of the seeds are then removed to obtain a clean section of the transverse section of the hypocotyl. After excision of the root tips, the explants were sectioned longitudinally between the original leaves. Place the two parts cut side up on a plant containing Murashige and mineral elements (Murashige et al. (1962) Physiol. Plant., 15: 473-497), Shepard's vitamin supplement (Shepard (1980) in Emergent Techniques for the Genetic Improvement of Crops (University of Minnesota Press, St. Paul, Minnesota), 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole- 3-acetic acid (IAA), 0.1 mg/l gibberellic acid (GA ₃ ), pH 5.6 and 8 g/l Phytagar on GBA medium.

颗粒加速器中通过置于样品2cm之上的150mm nytex屏轰击2次。Explants were subjected to microprojectile bombardment prior to Agrobacterium treatment (Bidney et al. (1992) Plant Mol. Biol. 18:301-313). This treatment was performed by placing 30 to 40 explant rings in the center of a 60 x 20 mm plate. Approximately 4.7 mg of 1.8 mm tungsten microprojectiles were suspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0), using 1.5 ml of liquid per bombardment. Each plate at PDS 1000

2 bombardments in a particle accelerator through a 150 mm nytex screen placed 2 cm above the sample.

The avirulent Agrobacterium tumefaciens strain EHA105 was used in all transformation experiments. A dual-purpose plasmid vector containing an expression cassette comprising the IPT gene linked to a ubiquitin promoter was introduced into Agrobacterium by the freeze-thaw method as described by Holsters et al. (1978) Mol. Gen. Genet. 163:181-187 strain EHA105. This plasmid further contains a kanamycin selectable marker gene (ie nptII). Bacteria for plant transformation experiments were grown overnight in liquid YEP medium (10 gm/l yeast extract, 10 gm/l bacto-peptone, 5 gm/l NaCl, pH 7.0) containing the bacteria and appropriate antibiotics required to maintain the dual-purpose plasmid (28°C, 100RPM continuous shaking). Suspensions with an _OD600 of 0.4 to 0.8 can be used. Agrobacterium cells were pelleted and resuspended to an OD600 of _0.5 in inoculation medium containing 12.5 mM MES pH 5.7, 1 gm/l _NH4Cl and 0.3 gm/l _MgSO4 .

Fresh bombarded explants were placed in the Agrobacterium suspension, mixed, and allowed to stand for 30 minutes. The explants were then transferred to GBA medium for co-cultivation at 26° C., 18-hours per day (light), cut side down. After 3 days of co-cultivation, the explants were transferred to 374B (GBA medium lacking growth regulators, sucrose level reduced to 1%) supplemented with 250 mg/l cefotaxime and 50 mg/l phosphate kanamycin. The explants were selected for 2 to 5 weeks and then transferred to fresh 374B medium without kanamycin for 1 to 2 weeks for continuous development. The growth areas with differentiated antibiotic resistance that did not produce shoots suitable for excision were transferred to GBA medium containing 250 mg/l cefotaxime for a second 3-day phytohormone treatment. Leaf samples from green kanamycin-resistant shoots were tested for the presence of NPTII by ELISA and for transgene expression by cytokinin synthesis activity. Such detection is described below.

Transplant NPTII-positive shoots into Pioneer Hybrid 6440, cultured sunflower seedling rhizomes in vitro. Surface sterilized seeds were germinated in 48-0 medium (half strength Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6) and cultured under the conditions described for explant culture. The upper part of the seedling was removed, a 1 cm vertical section was made on the hypocotyl, and the transformed shoot was inserted into the section. The whole section is wrapped with parafilm to protect the branches. Transplanted plants were transferred to soil for 1 week in vitro culture. The transplants in soil were kept under high humidity conditions and then slowly acclimatized to the greenhouse environment. Transformed fractions of _T0 plants (parental generation) matured in the greenhouse were identified by NPTII ELISA and/or analysis of leaf extracts for cytokinin synthesis activity, while transgenic seeds harvested from NPTII-positive _T0 plants were identified by analysis of a small fraction of The cytokinin synthesis activity of dry seed cotyledons was identified.

Example 16. IPT variants

A. Variant nucleotide sequences (SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76)

In SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45 , 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74, or 76 the nucleosides of ZmlPT1-9 or OsIPT1-11 The nucleotide sequence used to generate the nucleotide sequence of the open reading frame has 70%, 75%, 80%, 85%, 90% and 95% nucleotides when compared with the corresponding ORF starting unchanged nucleotide sequence Variant nucleotide sequences with acid sequence identity. These functional variants are generated by standard codon tables. When the nucleotide sequence of the variant is changed, the amino acid sequence encoded by its open reading frame is not changed.

B. Variant amino acid sequences of ZmlPT1-9 and OsIPT1-11

ZmlPT1-9 and OsIPT1-11 variant amino acid sequences were generated. In this example, one or more amino acids were changed. Specifically, the SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42 , 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 or 76 to determine the appropriate amino acid changes. Selected amino acid changes were made by reference to protein alignments with homologous proteins and other gene family members in various species. See Figure 1 and/or Figure 10. Selected amino acids that are not believed to be under high selective pressure (not highly conserved) can be replaced with considerable ease by amino acids with similar chemical characteristics (ie, same functional side chain). Functionality can be tested as described in Example 10. Can produce by this method and SEQ ID NO:1,3,4,5,7,8,10,11,13,14,16,17,19,20,21,22,24,26,28,40, Nucleotide sequence identity for 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74 and 76 A 70%, 75%, 80%, 85%, 90% or 95% variant.

B. Amino acid sequences of other variants of ZmlPT1-9 and OsIPT1-11

In this example, artificial protein sequences with 80%, 85%, 90% and 95% identity to the reference protein sequence were constructed. This requires the identification of conserved and variable regions in the alignments of Figure 1 and/or Figure 10, which are then constructed using amino acid substitution tables. This part will be described in detail below.

The determination of amino acid sequence changes is mainly based on the conserved regions of the IPT protein or other IPT polypeptides. Referring to Figures 1 and 10, based on the sequence alignment, the variable regions of the IPT polypeptides can be altered. Conservative substitutions in conserved regions are generally considered not to alter function. In addition, the skilled person should understand that the functional variants of the IPT sequence of the present invention may carry out a few non-conservative amino acid substitutions in the conserved region.

Artificial protein sequences that are 80-85%, 85-90%, 90-95% and 95-100% identical to the original sequence are then constructed. For example, to locate the midpoints of these intervals, plus or minus 1% degrees of freedom. Amino acid substitutions can be achieved through custom Perl scripts. The substitution table is shown in Table 6 below.

Table 8. Alternative table

amino acid Highly similar and preferred alternatives Changed sort order Notes I L, V 1 50:50 Alternative L I, V 2 50:50 Alternative V I, L 3 50:50 Alternative A G 4 G A 5 D E 6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 m L 17 The first methionine cannot be changed h Na no good alternative C Na no good alternative P Na no good alternative

Any conserved amino acids in the protein that should not be changed are first identified and removed from the substitution list. Starting methionine is automatically added to this list. Then make changes.

H, C and P cannot be changed. This change will first be made with isoleucine, across the N-terminus to the C-terminus. Then leucine and so on until the right target is reached. Intermediate numbers of substitutions can be made so as not to cause reverse changes. Listing order is 1-17, multiple isoleucine changes before leucine and methionine are necessary. And many amino acids do not need to be changed in this way. L, I and V participate in two optional preferred substitutions 50:50 substitutions.

Output the variant amino acid sequence. Calculate percent identity with a Perl script. With this program, the sequence corresponding to SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74, or 76 ORF start unchanged The amino acid identities of the nucleotide sequences were 82%, 87%, 92% and 97% for the ZmlPT1-9 and OsIPT1-11 variants.

Example 17. Rice IPT sequence characteristics

Eleven putative rice ipt sequences were identified that contained deduced amino acid sequences that shared similarity to Arabidopsis and petunia IPT proteins. Fig. 10 is an amino acid sequence alignment of Arabidopsis thaliana IPT protein (AtIPT), petunia IPT protein (Sho) and rice putative IPT protein (OsIPT). The asterisk indicates the amino acid position conserved in most IPT proteins, and its consensus sequence is GxTxxGK[ST]xxxxx[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG[ST](SEQ ID NO: 32) (wherein x represents any amino acid residue, [] represents any amino acid in [], x {m, n} m to n amino acid residue number) (Takei et al. (2001) J.Biol . Chem. 276:26405-26410).

The putative ATP/GTP-binding site (P-loop) element (prosite PS00017: consensus sequence [AG]-x(4)-G-K-[ST]), (SEQ ID NO: 69) is underlined. Amino acids 128-135 at SEQ ID NO:54; Amino acids 59-66 at SEQ ID NO:66; Amino acids 59-66 at SEQ ID NO:63; Amino acids 40-47 at SEQ ID NO:61; Amino acids 320-327 of ID NO: 43; Amino acids 22-29 of SEQ ID NO: 49; Amino acids 315-322 of SEQ ID NO: 59; Amino acids 32-39 of SEQ ID NO: 57; This domain is present at amino acids 41-48 of : 41; at amino acids 25-32 of SEQ ID NO: 46; and at amino acids 37-44 of SEQ ID NO: 52. The putative tRNA prenyltransferase domain (PF01715) is located at amino acids 59-348 of SEQ ID NO:57 and amino acids 69-352 of SEQ ID NO:41.

The overall amino acid sequence identities of the various rice IPT sequences compared to known Arabidopsis IPT sequences were determined using the Align X program with default settings. Table 9 summarizes these results. Table 10 lists polypeptides with homology to rice IPT sequences. This sequence was identified using BLASTP2.2.6.

Table 9

rice protein SEQ ID NO: Best IPT match Similarity (%) Identity (%) OsIPT7 54 AtIPT1 43.4 32.1 OsIPT6 57 AtIPT9 61 51.9 OsIPT10 59 AtIPT1 28 19.2 OsIPT3 63 AtIPT5 48.3 41.4 OsIPT8 41 AtIPT2 57.9 46.2 OsIPT11 43 AtIPT1 28.6 20.4 OsIPT4 66 AtIPT5 55.6 45.9 OsIPT5 52 AtIPT1 38.4 28.4 OsIPT1 49 AtIPT5 40.4 28.2 OsIPT9 61 AtIPT1 37.1 26.5 OsIPT2 46 AtIPT7 51.6 37.7

Table 10

Table 11. Summary Zm and Os IPT sequences

SEQ ID NO describe type 1 ZmIPT2 full length DNA 2 ZmIPT2 polypeptide AA 3 ZmIPT2 coding sequence DNA 76 Coding sequences of ZmlPT2 variants DNA 77 ZmIPT2 variant polypeptide AA 4 ZmIPT1 diploid sequence DNA 5 ZmIPT4 full length DNA 6 ZmIPT4 polypeptide AA 7 ZmIPT4 coding sequence DNA 8 ZmIPT5 full length DNA 9 ZmIPT5 polypeptide AA 10 ZmIPT5 coding sequence DNA 11 ZmIPT6 full length DNA 12 ZmIPT6 polypeptide AA 13 ZmIPT6 coding sequence DNA

SEQ ID NO describe type 14 ZmIPT7 full length DNA 15 ZmIPT7 polypeptide AA 16 ZmIPT7 coding sequence DNA 17 ZmIPT8 full length DNA 18 ZmIPT8 polypeptide AA 19 ZmIPT8 coding sequence DNA 20 ZmIPT9 full length DNA twenty one ZmIPT1 genome DNA twenty two ZmIPT1 full length DNA twenty three ZmIPT1 polypeptide AA twenty four ZmIPT1 coding sequence DNA 26 Variant ZmIPT1 Full Length DNA 27 Variant ZmIPT1 Peptide AA 28 Variant ZmlPT1 coding sequence DNA 25 ZmIPT1 promoter DNA 40 OsIPT8 genome DNA 41 OsIPT8 polypeptide AA 71 OsIPT8 coding sequence DNA 42 OsIPT11 genome DNA 43 OsIPT11 polypeptide AA 74 OsIPT11 coding sequence DNA 44 OsIPT2 genome DNA 45 OsIPT2 coding sequence DNA

SEQ ID NO describe type 46 OsIPT2 polypeptide AA 47 OsIPT1 genome DNA 48 OsIPT1 coding sequence DNA 49 OsIPT polypeptide AA 50 OsIPT5 genome DNA 51 OsIPT5 coding sequence DNA 52 OsIPT5 polypeptide AA 53 OsIPT7 genome DNA 54 OsIPT7 polypeptide AA 70 OsIPT7 coding sequence DNA 55 OsIPT6 genome DNA 56 OsIPT6 coding sequence DNA 57 OsIPT6 polypeptide AA 58 OsIPT10 genome DNA 59 OsIPT10 polypeptide AA 73 OsIPT10 coding sequence DNA 60 OsIPT9 genome DNA 61 OsIPT9 polypeptide AA 72 OsIPT9 coding sequence DNA 62 OsIPT3 genome DNA 63 OsIPT3 polypeptide AA 69 OsIPT3 coding sequence DNA

SEQ ID NO describe type 64 OsIPT4 genome DNA 65 OsIPT4 coding sequence DNA 66 OsIPT4 polypeptide AA 75 ZmIPT2 promoter DNA

Example 18.IPT Activity Detection

The ability of the protein encoded by the sequence of the present invention to synthesize cytokinin is detected in a bacterial culture medium. The results show that the sequence of the present invention encodes a protein with prenyltransferase activity. The reaction catalyzed by Agrobacterium ipt is shown in Akiyoshi et al. (1984) PNAS 81(19):5994-5998.

The IPT detection procedure was performed by the following reference: Kakimoto, T. (2001) Identification of plant biosynthetic enzymes as dimethylallyldiphosphate: ATP/ADP isopentenyltransferases, Plant Cell Physiol 42:677-685. Sakakibara, H., and Takei, K. (2002) Identification of Cytokinin Biosynthesis Genes in Arabidopsis: A Breakthrough for Understanding the Metabolic Pathway and the Regulation in Higher Plants, J. Plant Growth Regul. 21: 17-23. Sakano, Y., Okada, Y., Matsunaga, A., Suwama, T., Kaneko, T., Ito, K., Noguchi, H., and Abe, I. (2004) Molecular cloning, expression, and characterization Ofadenylate isopentenyltransferase from hop (Humulus lupulus L.), Phytochemistry 65: 2439-2446.

The ZmlPT2 gene was amplified with gene-specific primers with appropriate NdeI and NotI restriction site extensions and cloned into pET28a (N-terminal tag) or pET30b (C-terminal tag) digested with NdeI and NotI. The sequence of the resulting plasmids was verified by sequencing the translation of the His tag fused to ZmlPT2, and BL21-Star ^™ E. coli competent cells (Invitrogen ^™ ) were transformed with pET28a-ZmlPT2 and pET30b-ZmlPT2. Similarly, the tzs IPT gene derived from Agrobacterium tumefaciens was cloned into pET28a to obtain the plasmid Rosetta2(DE3)pLysS for transformation.

Recombinant his-tagged proteins were purified using TALON ^™ columns (BD Biosciences) according to the manufacturer's instructions. Dimethylallyl diphosphate (DMAPP)::AMP and DMAPP::ATP prenyltransferase activities were assayed using purified protein by the following procedure:

● React the purified protein extract in a reaction mixture containing 12.5 mM Tris-HCl (pH 7.5), 37.5 mM KCl, 5 mM MgCl ₂ , 1 mM DMAPP and 1 mM AMP or ATP at 30° C. for 2 hours. The reaction was terminated by boiling the sample for 5 minutes.

Treat half of the reaction mixture with calf intestinal alkaline phosphatase (CAIP), add 1 volume of 2x CAIP reaction buffer (0.45M Tris-HCl pH 9, 10mM MgCl ₂ , 1000 units CAIP/ml) at 37°C Incubate for 1 hour.

Separation of the reaction products by reverse phase HPLC (Agilent 1100 system with diode array detector) over a C18-ODS2 column (Phenomenex) with 0.1M acetic acid, pH 3.3 (Buffer A) and acetonitrile (Buffer B) by the following procedure Separation:

o 100% buffer A elution for 15 minutes.

o Elution with a linear gradient from 100% Buffer A and 0% Buffer B to 20% Buffer A and 80% Buffer B over 35 minutes.

UV absorbance was monitored at 280nm. Product retention times were compared to standards obtained from Sigma or OlChemIm.

Firstly, recombinant Tzs and ZmIPT2 proteins were used to measure DMAPP::AMP prenyltransferase activity. Figures 12A and 12B show the HPLC chromatograms of one substrate of the reaction, 5'-AMP (Sigma), and the expected product, isopentenyladenosine 5'-monophosphate (iPMP) (OlChemIm). Chromatograms obtained with the IPT(tzs) protein showed conversion of almost all 5'-AMP substrates to iPMPs (Fig. 12C). Likewise, chromatograms obtained with ZmIPT2 purified protein showed that the enzyme was able to convert 5'-AMP to iPMP, but less efficiently than Agrobacterium IPT because not all 5'-AMP was converted (Fig. 12D).

The reaction product treated with calf intestinal alkaline phosphatase (CAIP) and the HPLC chromatogram showed the identity of the reaction product iPMP. Figures 13A and 13B show chromatograms obtained with adenosine (Ado) (Sigma) and isopentenyladenosine (iPAR) (Sigma). As expected, after dephosphorylation of the products of each reaction, iPMP was converted to isopentenyladenosine (iPAR) (Figures 13C and 13D), where the remaining 5'-AMP was converted to Ado (Figure 13D). This suggests that ZmlPT2 can metabolize 5'-AMP and DMAPP to iPMP.

DMAPP::ATP activity was determined by the same reaction buffer, but replacing 5'-AMP with 5'-ATP in the reaction mixture. Figure 14A shows the chromatogram obtained with 5'-ATP. If ZmlPT2 can catalyze the transfer of DMAPP to 5'ATP, the resulting product can generate iPTP. The chromatogram in Figure 14B shows that all 5'-ATP is metabolized to iPTP by ZmlPT2, indicating that ZmlPT2 uses 5'-ATP more efficiently than 5'-AMP. The reaction product was treated with CAIP to confirm its identity. This processing should result in iPAR. After separation by HPLC, the chromatograms were compared to iPAR standard chromatograms (Figure 14C). After treatment, the reaction product was converted into iPAR (Fig. 14D), thus indicating that ZmlPT2 can metabolize 5'-ATP and DMAPP to iPTP. Together these results demonstrate that ZmlPT2 is a cytokinin synthase that preferentially uses 5'-ATP as a substrate.

The same experiments showed that 5'-ADP is also a suitable substrate for the encoded enzyme. Taken together, these results demonstrate that ZmlPT2 is a cytokinin synthase that preferentially uses 5'-ATP as a substrate. Future experiments will determine the kinetic properties of each substrate with purified ZmlPT2 protein.

Example 19. Detection of ZmlPT2 protein in developing grain

To further study the expression profile of ZmlPT2 protein, Western blotting experiments were performed using polyclonal antibodies. Polyclonal antibodies against purified N-terminal His-tagged recombinant ZmlPT2 protein were obtained from rabbits. Fifteen micrograms of protein extracted from whole grains harvested at different days after pollination (DAP) were subjected to SDS-PAGE and transferred to PVDF membranes. The ZmlPT2 protein was detected by the method of Laemmli (Nature 227:680-685, 1970) with the anti-ZmlPT2 polyclonal antibody as the primary antibody and the goat anti-rabbit IgG antibody coupled with alkaline phosphatase as the secondary antibody.

Figure 15 shows the ZmIPT2 protein level increased from 0 to 10 DAP, peaked at 10 DAP, then decreased from 10 DAP to 15 DAP, and stabilized at a constant level thereafter. This is consistent with the results shown by Northern blot that the gene expression peaked at 10 DAP and strongly expressed in the pedicel and endosperm. Although the overall gene expression decreased thereafter, the expression level in the pedicel remained high in the later stage. ZmIPT2 protein levels are very high in grains compared to other organs. The results suggest that the cytokinin activity of ZmlPT2 protein in grains is more likely to be regulated at the transcriptional level. Western blot analysis of protein levels in grains indicated that the antibody was very specific for ZmlPT2. Antibodies and in situ hybridization are very useful in determining the precise site of gene expression during the grain response phase.

Example 20. Abnormal overexpression of ZmlPT2 in transgenic Arabidopsis

The previous examples described the overexpression of ZmlPT2 in Arabidopsis callus. To study the effects of overexpression of ZmlPT2 at the whole plant level, Arabidopsis plants were transformed with A. tumefaciens strains containing plasmids including the construct 35S-AdhI-ZmlPT2-PiuII, marker for the bar herbicide resistance gene. (Thompson et al. (1987) EMBO J 6 (9): 2519-2523; White et al. (1990) Nucleic Acids Res. 18 (4): 1062) using a simplified Arabidopsis transformation procedure (Clough and Bent ( 1998) Plant J. 16:735-743). Seeds were planted on flat soil containing soil and incubated for 2 days at 4°C to optimize germination. After 10 days of cultivation in the greenhouse, transformants were selected by spraying the seedlings daily with 1/1000 dilution of Finale ^™ herbicide for 5 days.

After screening, plants that are tolerant to the herbicide treatment are identified. Some plants are smaller than others and are dark green. The green color of leaves is associated with cytokinins, indicating that the dark green transformed plants have increased levels of cytokinins. Some transgenic plants were more affected than others, possibly related to the expression levels of transgenes known to vary depending on positional effects associated with the insertion in the genome.

At an early stage of development, certain transgenic plants showed accumulation of anthocyanins in leaves compared to non-transgenic plants. Certain transgenic plants had highly serrated leaves compared to wild-type Arabidopsis. This phenotype has been reported in Arabidopsis plants overexpressing the Agrobacterium ipt gene (van der Graaff et al. (2001) Plant Growth Regul. 34(3):305-315). High levels of cytokinins are often detrimental to plant growth (van der Graaff et al., 2001), and some transgenic plants struggled during development compared to control plants. Certain transgenic plants have reduced apical dominance in inflorescence stems compared to controls, as has been reported in Arabidopsis and tobacco plants containing high levels of cytokinins (van der Graaff et al., 2001; Crozier et al. ( 2000) Biosynthesis of hormones andellicitor molecules. In Biochemistry and molecular biology of plants, B. Buchanan, W. Gruissem, and R.L. Jones, eds (Rockville, Maryland: American Society of Plant Biologists), pp. 850-929).

Certain transgenic plants had a "branching" phenotype, probably due to reduced apical dominance resulting in a large number of leaves. Certain plants had poor seed fixation due to lack of siliques containing few seeds or smaller siliques. They also exhibit anthocyanin accumulation in leaves and along inflorescence stems. Plants usually exhibit serrated cauline leaves. The most extreme phenotypes were transgenic plants with rosettes (leaves) approximately 5 mm in diameter, with very small curled leaves signaling anthocyanin accumulation. The plant produces flowers but does not produce seeds. It also exhibits an unusually large number of tufts of large fragrant hairs. The curled leaf phenotype was previously described in tobacco with high levels of cytokinins (Crozier et al., 2000).

Thus, the overexpression of the protein in Arabidopsis thaliana further suggests that the protein's function is consistent with previous overexpression of the IPT gene in Arabidopsis and tobacco by constructing various phenotypes (Van der Graaff at al., 2001; Crozier et al., 2000). The phenotypes observed in several independent transgenic plants were consistent with cytokinin accumulation levels, suggesting that ZmlPT2 is a cytokinin synthase.

Example 21. Determination of Gene Function by Mu Tags

Gene function can be further verified by studying mutants that disrupt transcription and/or translation of the target sequence. In a specific technical solution, it is carried out by the method described in US 5,962,764. The Trait Utility System for Corn (TUSC) is a private resource for screening gene-specific transposon insertions from saturated maize mutants via the Mutator transposable element system. For example, the effect of the ZmlPT sequences of the invention on traits such as plant pool strength can be studied. TUSC mutants of ZmlPT2 were identified by the following method.

A 1495bp gene sequence was provided for TUSC screening to identify seedling Mutator insertions in the maize IPT2 gene (ZmIPT2). The gene revealed by the work contains a 966 bp open reading frame (ORF; nt83-1048) not disrupted by introns.

Initial screening of the TUSC DNA library was performed with two ZmlPT2-specific primers (PHN79087 and PHN79088), each in combination with a Mutator terminal inverted repeat (TIR) primer, as described in US 5,962,764. The primer sequences are listed below, and the sequence numbers are respectively SEQ ID Nos: 82-84.

PHN79087 zmIPT2-F 5′＞TGTTGTGTGCACAGAATCGAGCGG＜3′

PHN79088 zmIPT2-R 5′＞CGTCCGCTAGCTACTTATGCATCAG＜3′

PHN9242 MuTIR 5′＞AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC＜3′

The primers were verified by gene-specific amplification of ZmIPT2 by combining B73 genomic DNA with PHN79087+79088 primers before use. Control amplification products were excised from agarose gels and used as ³² P-labeled hybridization probes for TUSC screening.

Primer pair Expected (re. reference sequence) (bp) Observed B73gDNA (bp) 79087+79088 1033 ~1050

After successive rounds of screening of TUSC DNA template pools and individual samples by PCR and ZmlPT2 hybridization, the heritability of the expected zmlPT2::Mu allele was tested across germplasm. Detection was performed by repeated ZmlPT2::Mu PCR on DNA templates from 5 grains from selfed (F2) seeds of a single TUSC plant screened. One of the TUSC families, PV03_13 H-07 (from library No. 26), showed strong positive results for both 79087+9242 and 79088+9242 primer combinations in F2 template detection. These PCR products were cloned in Topo-TA vector (Invitrogen) to verify the DNA sequence of the zmlPT2Mu insertion allele contained in the PV03 13H-07 family. Each PCR fragment is predicted to be homologous to the ZmlPT2 locus and also has ~71 bp of Mutator TIR homology. The expected 9 bp host site duplication resulting from the insertion of the Mu element into the maize genomic DNA is also expected for the PV03 13H-07 ZmlPT2::Mu allele.

As shown in Figure 16, the DNA sequences of each TUSC CPR product exhibited these expected features. The 79087+9242 PCR product contains a 600bp fragment directly homologous to ZmlPT2 on the left side of the PV03 13H-07 Mu insertion site. The 79088+9242 PCR product contains a 442bp fragment identical to the DNA sequence of ZmlPT2, which indicates the right side of the Mu insertion site and contains the PHN79088 primer site. The Mutator TIR sequence was processed and compared with the zmlPT2 reference sequence. These PCR fragments overlapped by 9 bp (nucleotides 624-632 in the 1495 bp ZmlPT2 reference sequence), indicating that the insertion of Mutator into ZmlPT2 produced the expected 9 bp host site duplication.

Therefore, PV03 13H-07 of the TUSC family contains a heritable Mutator insertion in the coding sequence (ORF) of the ZmlPT2 gene. This allele is expected to produce a null mutation or "knockout" of the ZmlPT2 locus. F2 progeny seeds from PV03 13H-07 genetically isolated with ZmIPT2::Mu mutation, zmIPT2-H07, were isolated and propagated from the TUSC seed bank for phenotypic and molecular biological analyses.

Figure 16 graphically summarizes the TUSC results and the corresponding sequence is SEQ ID NO:85.

As an additional feature, a BLAST search was performed on the MuTIR portion of each zmlPT2::Mu PCR product to summarize the identity of the Mu element located at the ZmlPT2 locus. The TUSC PCR product amplified with Mutator PHN9242 primers contained 39 bp TIR flanking sequences targeting the amplified intrinsic element. BLAST results were consistent with the zmlIPT2::Mu element for Mu4 or Mu3 elements.

>IPT2_TIR_L (SEQ ID NO: 86)

GAGATAATTGCCATTATAGAAGAAGAGAGAAGGGGATTCGACGAAATAGAGGCGATGGCGTTGGCTTCTCT

>IPT2_TIR_R (SEQ ID NO: 87)

AAGCCAACGCCAACGCCTCTATTTCGTCGAATCCCCTTCTCTCTTCTTCTATAATGGCAATTATCTC

In certain embodiments, the nucleic acid constructs of the invention can be combined ("stacked") with other nucleotide sequences of interest to produce plants with the desired phenotype. The polynucleotides of the invention can be stacked with any gene or combination of genes, and the resulting combination can include multiple copies of any one or more polynucleotides of interest. Desired combinations can affect one or more traits; that is, specific combinations can be created to modulate the expression of genes that affect cytokinin activity. For example, upregulation of cytokinin synthesis can be coupled with downregulation of cytokinin degradation. Other combinations can be designed to produce plants having various desired traits, such as those described previously.

All documents and patent applications related to the present invention are indicative for those skilled in the art. All literature and patent applications are herein incorporated by reference to the same extent that each individual literature or patent application is specifically and individually indicated to be incorporated herein by reference.

While the foregoing invention has been described in detail by way of example and embodiment for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

sequence listing

<110>Pioneer Hi-Bred International, Inc.

<120> Prenyltransferase sequences and methods of use thereof

<130>1507-AR

<150>60/610,656

<151>2004-09-17

<150>60/637,230

<151>2004-12-17

<150>60/696,405

<151>2005-07-01

<160>87

<170>FastSEQ for Windows Version 4.0

<210>1

<211>1495

<212>DNA

<213> Corn (Zea mays)

<220>

<221> CDS

<222>(83)...(1051)

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT2 full-length sequence

<400>1

aaaaggcacg gactgcttct ttttctattt tttgttgtgt gcacagaatc gagcggctac 60

aataatcaag atcatcaaga ca atg gag cac ggt gcc gtc gcc ggg aag ccc 112

                                                                                        , 

1 5 10

aag gtg gtg ttc gtg ctc ggc gcc aca gcg aca ggg aag tcg aag ctc 160

Lys Val Val Phe Val Leu Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu

15 20 25

gcc atc gcc ctc gcc gag cgc ttc aac ggt gag gtt atc aac gct gac 208

Ala Ile Ala Leu Ala Glu Arg Phe Asn Gly Glu Val Ile Asn Ala Asp

30 35 40

aaa atc cag gtc cac gat ggc gtg ccc atc atc acg aac aag gtc aca 256

Lys Ile Gln Val His Asp Gly Val Pro Ile Ile Thr Asn Lys Val Thr

45 50 55

gag gaa gag cag ggc ggg gtg ccc cac cac ctg ctc agc gtc cgc cac 304

Glu Glu Glu Gln Gly Gly Val Pro His His Leu Leu Ser Val Arg His

60 65 70

ccg gac gcc gac ttc act gcg gag gag ttc cga cgt gag gcg gcc agc 352

Pro Asp Ala Asp Phe Thr Ala Glu Glu Phe Arg Arg Glu Ala Ala Ser

75 80 85 90

gcc gtg gcc cgc gtg ctc tcg gcg ggc cgc ctc ccc gtc gtg gca ggc 400

Ala Val Ala Arg Val Leu Ser Ala Gly Arg Leu Pro Val Val Ala Gly

95 100 105

ggg tcc aac acc tac atc gag gca ctg gtg gaa ggc gac ggc gcc gcc 448

Gly Ser Asn Thr Tyr Ile Glu Ala Leu Val Glu Gly Asp Gly Ala Ala

110 115 120

ttc cgc gcg gcg cac gac ctc ctc ttc gtc tgg gtg gac gcg gag cag 496

Phe Arg Ala Ala His Asp Leu Leu Phe Val Trp Val Asp Ala Glu Gln

125 130 135

gag ctg ctg gag tgg tac gcc gcg ctg cgc gtg gac gag atg gtg gcc 544

Glu Leu Leu Glu Trp Tyr Ala Ala Leu Arg Val Asp Glu Met Val Ala

140 145 150

cgc ggg ctg gtg agc gag gct cgc gcg gcg ttc ggc ggc gcc ggg gtt 592

Arg Gly Leu Val Ser Glu Ala Arg Ala Ala Phe Gly Gly Ala Gly Val

155 160 165 170

gac tac aac cat ggc gtg cgc cgc gcc atc ggc ctg ccg gag atg cac 640

Asp Tyr Asn His Gly Val Arg Arg Ala Ile Gly Leu Pro Glu Met His

175 180 185

gcc tac ctg gtg gcg gag cgc gag ggc gtc gct ggg gag gcc gag ctc 688

Ala Tyr Leu Val Ala Glu Arg Glu Gly Val Ala Gly Glu Ala Glu Leu

190 195 200

gcg gcc atg ctg gaa cgc gcg gtg cgc gag atc aag gac aac acc ttc 736

Ala Ala Met Leu Glu Arg Ala Val Arg Glu Ile Lys Asp Asn Thr Phe

205 210 215

cgc ctc gcg cgc acg cag gcg gag aag atc cgg cgc ctc agc acg ctc 784

Arg Leu Ala Arg Thr Gln Ala Glu Lys Ile Arg Arg Leu Ser Thr Leu

220 225 230

gac ggc tgg gac gtc cgc cgc atc gac gtg acc ccc gtg ttc gcg cgc 832

Asp Gly Trp Asp Val Arg Arg Ile Asp Val Thr Pro Val Phe Ala Arg

235 240 245 250

aag gcc gat ggc act gag tgc cac gag ctg act tgg aag aag cag gtg 880

Lys Ala Asp Gly Thr Glu Cys His Glu Leu Thr Trp Lys Lys Gln Val

255 260 265

tgg gag ccg tgc gag gag atg gtg agg gct ttc ctc gag ccg tcc ctg 928

Trp Glu Pro Cys Glu Glu Met Val Arg Ala Phe Leu Glu Pro Ser Leu

270 275 280

act gcc gtt cca ggt gtt gca gta act gaa gaa ggg aac gcc ggc gtc 976

Thr Ala Val Pro Gly Val Ala Val Thr Glu Glu Gly Asn Ala Gly Val

285 290 295

gtc gct act gct gca ccc gct ggt gat gtc gtc gtc cca act ggc gat 1024

Val Ala Thr Ala Ala Pro Ala Gly Asp Val Val Val Pro Thr Gly Asp

300 305 310

gtc gtc acc gcc gtg gct gat gca taa gtagctagcg gacgtagcgc 1071

Val Val Thr Ala Val Ala Asp Ala *

315 320

atgcatgcaa tgcatgcagg ctggctggct ggcttaatta gtgcctccga cttgctttaa 1131

actcatgtag ctgcgtccat gggagagggt gagatacaag tttatgcgac ttatatttct 1191

ttctaaattt aaatggatct cggatccgta gtatctggtt taatataatt ataatatttc 1251

cttcgaatta ttatatatat atgctcacac tcagttaggg atatatactc cctccattca 1311

ctctatgtat ttggattcat atgcaaaagt attttaaaat tatactacct ccattctcga 1371

atatttgtta cccgcttgtt tattttctaa aacatgataa ataaaaaaac ggagagaata 1431

gtattttatt atttgttgat gatatatttt gtaagatatg aacggtgaaa gttttaccat 1491

aaag 1495

<210>2

<211>322

<212>PRT

<213> corn

<400>2

Met Glu His Gly Ala Val Ala Gly Lys Pro Lys Val Val Phe Val Leu

1 5 10 15

Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu Ala Ile Ala Leu Ala Glu

20 25 30

Arg Phe Asn Gly Glu Val Ile Asn Ala Asp Lys Ile Gln Val His Asp

35 40 45

Gly Val Pro Ile Ile Thr Asn Lys Val Thr Glu Glu Glu Gln Gly Gly

50 55 60

Val Pro His His Leu Leu Ser Val Arg His Pro Asp Ala Asp Phe Thr

65 70 75 80

Ala Glu Glu Phe Arg Arg Glu Ala Ala Ser Ala Val Ala Arg Val Leu

85 90 95

Ser Ala Gly Arg Leu Pro Val Val Ala Gly Gly Ser Asn Thr Tyr Ile

100 105 110

Glu Ala Leu Val Glu Gly Asp Gly Ala Ala Phe Arg Ala Ala His Asp

115 120 125

Leu Leu Phe Val Trp Val Asp Ala Glu Gln Glu Leu Leu Glu Trp Tyr

130 135 140

Ala Ala Leu Arg Val Asp Glu Met Val Ala Arg Gly Leu Val Ser Glu

145 150 155 160

Ala Arg Ala Ala Phe Gly Gly Ala Gly Val Asp Tyr Asn His Gly Val

165 170 175

Arg Arg Ala Ile Gly Leu Pro Glu Met His Ala Tyr Leu Val Ala Glu

180 185 190

Arg Glu Gly Val Ala Gly Glu Ala Glu Leu Ala Ala Met Leu Glu Arg

195 200 205

Ala Val Arg Glu Ile Lys Asp Asn Thr Phe Arg Leu Ala Arg Thr Gln

210 215 220

Ala Glu Lys Ile Arg Arg Leu Ser Thr Leu Asp Gly Trp Asp Val Arg

225 230 235 240

Arg Ile Asp Val Thr Pro Val Phe Ala Arg Lys Ala Asp Gly Thr Glu

245 250 255

Cys His Glu Leu Thr Trp Lys Lys Gln Val Trp Glu Pro Cys Glu Glu

260 265 270

Met Val Arg Ala Phe Leu Glu Pro Ser Leu Thr Ala Val Pro Gly Val

275 280 285

Ala Val Thr Glu Glu Gly Asn Ala Gly Val Val Ala Thr Ala Ala Pro

290 295 300

Ala Gly Asp Val Val Val Pro Thr Gly Asp Val Val Thr Ala Val Ala

305 310 315 320

Asp Ala

<210>3

<211>969

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT2 coding sequence

<400>3

atggagcacg gtgccgtcgc cgggaagccc aaggtggtgt tcgtgctcgg cgccacagcg 60

acagggaagt cgaagctcgc catcgccctc gccgagcgct tcaacggtga ggttatcaac 120

gctgacaaaa tccaggtcca cgatggcgtg cccatcatca cgaacaaggt cacagaggaa 180

gagcagggcg gggtgcccca ccacctgctc agcgtccgcc acccggacgc cgacttcact 240

gcggaggagt tccgacgtga ggcggccagc gccgtggccc gcgtgctctc ggcgggccgc 300

ctccccgtcg tggcaggcgg gtccaacacc tacatcgagg cactggtgga aggcgacggc 360

gccgccttcc gcgcggcgca cgacctcctc ttcgtctggg tggacgcgga gcaggagctg 420

ctggagtggt acgccgcgct gcgcgtggac gagatggtgg cccgcgggct ggtgagcgag 480

gctcgcgcgg cgttcggcgg cgccggggtt gactacaacc atggcgtgcg ccgcgccatc 540

ggcctgccgg agatgcacgc ctacctggtg gcggagcgcg agggcgtcgc tggggaggcc 600

gagctcgcgg ccatgctgga acgcgcggtg cgcgagatca aggacaacac cttccgcctc 660

gcgcgcacgc aggcggagaa gatccggcgc ctcagcacgc tcgacggctg ggacgtccgc 720

cgcatcgacg tgacccccgt gttcgcgcgc aaggccgatg gcactgagtg ccacgagctg 780

acttggaaga agcaggtgtg ggagccgtgc gaggagatgg tgagggcttt cctcgagccg 840

tccctgactg ccgttccagg tgttgcagta actgaagaag ggaacgccgg cgtcgtcgct 900

actgctgcac ccgctggtga tgtcgtcgtc ccaactggcg atgtcgtcac cgccgtggct 960

gatgcataa 969

<210>4

<211>2901

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT1 partial sequence

<400>4

gccgactatt tgttagtcag ctaactatta gctctagtaa attcaaatgg agtctaagct 60

cttagttgtg ctatggttgt attttatcaa taattataac taaatttggc aagtgtttat 120

taattgagcg tctagattcc atgtgattca tgtgataaag catgccaatt aggccccgtt 180

tgactcctct tacaaaagtt tagcactaca tttttttggt aaagtttagc tactaaagaa 240

acaacagaat tcctataagt ggtgtaccaa aatttagcat tcattagcat taccaaagat 300

gctaaactat attaaaaata gtcccctaga accatttctc actccccctc cccaccaaat 360

tcttctctct ctctctcctc ccacaactga ccgctagctt ctgctgtcac gcaggtgacg 420

ctagcttcca cattaattag ggataatatg ttaaacactt gcatcaaaca tgtctcaagt 480

tctaaagttt aaccaaagtt taagcccatt ttagaaacca cagtttttaa aatactataa 540

tatactttag tcattacaat accacagttt gaaataatac agtttttact ttagtaatta 600

caataccaca gtttgaaata atacagtttt acaaactgtg gtcaatacag ttttgcaaac 660

taaggtccag acctaagttt agaatagctt aaaataacta cagtatttgc aatacttcgg 720

ttttgaaaat agagatttta gcaatcttaa acacctcctt agcatatgct aaactttagc 780

agtatagaaa gatggaccat tgttcaagta tctatccact tccacactac gtttctgtgg 840

cttctagacc cgtcgtacgt gtggtacaat acttttgata agcagttctt tggtgcctgc 900

ttcttccgct gctaaaaggc atggaggtag tccccccgtc gacgtgccag gaattcggga 960

ttgggaaagc atgtcgctgt caggctggca cgcttttttg ggtgggacga gacgaccatg 1020

tccacgtcaa cgtccccgtc ccacttctcg ccggttctgc tgtgaaaaag ttaccatcag 1080

ctgcggtctt ggggcagcag cgatggccat ggcctcaggc tcttctgctt caggcgctgc 1140

actcgcttcc ggtggcctcg cccttgcctg tgctgctccc actcgtgcgc acctcgcagc 1200

acccggttgc cgcaggcctg tacatccgca tgcatgcttt tcttatgtct gcttttcctt 1260

cctatgcaga cagtacagct ctcagaaaga aggaaaaaaa actagactca cctcgcacac 1320

atattgcgtc cacagctctc ttgaagaggg gtcacttgcg cttgtgcttg gcaaaacagt 1380

ggaattgtcc gacaaaaacc ttcttacgac atccacacat ggtttgacaa ctttcttttg 1440

ccatgagtcg tcagtggcac ctatcaaaat atgagaggga gattatgagt tgctacagag 1500

aaggcatacg catctgacag ggtaccaatg gacttacaga agaatgcttc ggttgcgtca 1560

acacgatgca agttccacccc aaaatcttta ctcagccgat gcaatctccg tctcttttat 1620

ttaatggaga aaaaaaaaga aacgctaaga agtctacatt gagccaagta tccgtgtgtt 1680

cagtcgcaca gtattctaaa acaacacaac aacataaaaa aatacacagg atactaagtg 1740

ccttacttgac gtcgaactag tctacgagtg tttgccttca gctgggaaac agcttcgtcc 1800

aacaagctct taagctgatc gtcatgcata cctaacaggc ttgcacagga accctcacca 1860

gattcttttc tgggtaaata tgctctgaaa aactcgtcaa actcacgaac cccaatagcc 1920

tgccgcagcc cctgggtata gacagcatcc gcatcatata tgctgcatac ttcgtccagc 1980

aggccaccat ccatcatgca atcgaccctt ttgttgacat aactgtccag gacttgaaga 2040

tcagcatcta cccacaggaa acagcagtcg agtctggagt tactaggccg accccatttc 2100

tgttctgaca tgatgagcag gaattcccag caagtttata ctcaattagg gaaacaaaac 2160

caaaaaaaga agagccttgt gaggcaattg tcggctcatg ttcatgacaa agggaggata 2220

ttatacaagc agagcagaca agtgcagcga aaaattcctc accttagcgg cctctccttg 2280

gaacagatcg ctgggtaggg cacccgtggt tgcatacaac tcgaggtagc gtttgatctt 2340

tgtgatacac caacccatgc agaacgaaaa atacacaagc aatcaacggc agtaggaaaa 2400

tgagcacctg tacaagaagg aattctaaca tggtagagct caagagctgc ataaagtcag 2460

gaaaaacttg ggcaacaccc ttacttttct atggtcgttt ggatggatcc tctgcgcagc 2520

cacaggatcg atctccttca agcgttcaaa cccattgcct tcatcttcat cagtaagacc 2580

tgacgattgg catacatgtc aaccgctgat tgttcatact aagaacaagg aacatttagc 2640

atgactcatt attcgcctat accatcatct atgtgatctc tcagagtaca gccctgcatt 2700

tcttctgcca tatcatccaa gaggaatggg ctaacgagag cctggagtat tttgtgggca 2760

aaaacacacg gtggttcaga cactgcggtc accccagaaaa ccatacagaa atcaggacac 2820

ttcaaatttg aacctggatg tagaagtttg tgccgccgac aaccacaggg aggccaccgc 2880

ggtccactat ttcctgtata a 2901

<210>5

<211>2654

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT4 full length

<221> CDS

<222>(904)...(1998)

<400>5

ttttggggga tcccaaagaa actagtatgt ggattgctcg tcgccgcgct gcacacatgc 60

ttcgtgcgct gcaaaatctg tccacgcgcg cgggcgcaca cacattattt tttcgttcat 120

ttgtatctgt gaaagatacg tacaaaatct gcgtgtatct tccatgattt gacaagaatt 180

tcagacacca aggcaaattt tgttggtaca tacagctcag cagcagtcct ttctgttgac 240

ctgctgcccc cttttgcttc cgttctcgat ccaaatctat ggttgaatct ttcttctgat 300

caccaggatt gttgttgttt agactttaga tcatcctgat cctgcgtgtc cggatcaaat 360

ctgcaaatcc aacgcaaaaa aaatggttca cgtgctaggt acagtttctc aaacatcaca 420

tatttggtcg tttatggtct aatgccaatt tcaaaatggg agatttcgtt ttcctcaaat 480

cgtacaagaa tgaagctgcc atatagtacg aaattcctct gaaattccag agcttcgaat 540

tacacaatcc aattaattgg ctacatgaag gtagcgccgt agcggccgac gtaccattcc 600

gacgacccca tcaatataat gggcatgggg gggcatctga atctttatct aaaatacatt 660

tgttttcgtt tcaatctaat ctaaccccaa attaaaggaa cgcatttgca catttttgta 720

ttgcaacgag gaggatccat ttgcccactt gttttcgcca gcacaagata aatagcagcc 780

acacccagca catcgacctg cagccaactc acgaactaac taccagaaga tctcaaaggc 840

ttcgattctt gaccccaagc aagtggttaa gcataagcaa cttaaacgac agcgacagaa 900

acc atg tca ctc tac ttg gcg ccc acg gcc gcc gct gcc acc acc acc 948

Met Ser Leu Tyr Leu Ala Pro Thr Ala Ala Ala Ala Thr Thr Thr

1 5 10 15

acc acc acc ctc cca agg cag ctg cta cca gcg ccg tcc atc gac cta 996

Thr Thr Thr Leu Pro Arg Gln Leu Leu Pro Ala Pro Ser Ile Asp Leu

20 25 30

agg cgg ctg gaa cga cgt ggc atg gcg ctg ccc ctg cca ccg gcg ccg 1044

Arg Arg Leu Glu Arg Arg Gly Met Ala Leu Pro Leu Pro Pro Ala Pro

35 40 45

gcg ccg ccg cca ccg ctg gtc agt gcc aac aac agg cat gcc gga gcg 1092

Ala Pro Pro Pro Pro Leu Val Ser Ala Asn Asn Arg His Ala Gly Ala

50 55 60

aag cac aag gcc gtg gtg gtg atg ggc gcc acg ggg acc ggc aag tcg 1140

Lys His Lys Ala Val Val Val Met Gly Ala Thr Gly Thr Gly Lys Ser

65 70 75

cgg ctg gcg gtg gac ctg gcg ctc cgg ttc ggc ggc gag gtc atc aac 1188

Arg Leu Ala Val Asp Leu Ala Leu Arg Phe Gly Gly Glu Val Ile Asn

80 85 90 95

tcg gac aag atc cag ctg cac gcc ggc ctg gac gtg acc ag aac aag 1236

Ser Asp Lys Ile Gln Leu His Ala Gly Leu Asp Val Thr Thr Asn Lys

100 105 110

gtg acc gag cag gag cgc gcc ggc gtg ccg cac cac ctg ctc ggg gtg 1284

Val Thr Glu Gln Glu Arg Ala Gly Val Pro His His Leu Leu Gly Val

115 120 125

gcg cgg ccc gac gag gag ttc acg gcc gcg gac ttc cgg cgc gag gcg 1332

Ala Arg Pro Asp Glu Glu Phe Thr Ala Ala Asp Phe Arg Arg Glu Ala

130 135 140

acc cgc gcc gcg cgc gcc atc acc gcg cgc ggc cgc ctg ccc atc gtc 1380

Thr Arg Ala Ala Arg Ala Ile Thr Ala Arg Gly Arg Leu Pro Ile Val

145 150 155

gcg gga ggg tcc aac tcg tac gtg gag gag ctg gtg gac ggc gac cgc 1428

Ala Gly Gly Ser Asn Ser Tyr Val Glu Glu Leu Val Asp Gly Asp Arg

160 165 170 175

gcc gcg ttc cgc gac cgc tac gac tgc tgc ttc ctc tgg gtg gac gtg 1476

Ala Ala Phe Arg Asp Arg Tyr Asp Cys Cys Phe Leu Trp Val Asp Val

180 185 190

cag cgc gcc gtg ctg cac ggc tgc gtg gcg cgg cgc gtc gac gag atg 1524

Gln Arg Ala Val Leu His Gly Cys Val Ala Arg Arg Val Asp Glu Met

195 200 205

cgc gcc cgc ggc ctg gtg gac gag gtc gcg gcg gcc ttc gac ccg cgc 1572

Arg Ala Arg Gly Leu Val Asp Glu Val Ala Ala Ala Phe Asp Pro Arg

210 215 220

cgc aac gac tac tcg cgc ggc ctc tgg cgc gcc att ggc gcg ccc gag 1620

Arg Asn Asp Tyr Ser Arg Gly Leu Trp Arg Ala Ile Gly Ala Pro Glu

225 230 235

ctc gac gcg tac ctg cgg tgg ccg gga ccg ggt gta gac ggc gac gcg 1668

Leu Asp Ala Tyr Leu Arg Trp Pro Gly Pro Gly Val Asp Gly Asp Ala

240 245 250 255

gaa agc gag ggc gag cgc gac cgg ctg ctg gcc gcc gcc atc gag gac 1716

Glu Ser Glu Gly Glu Arg Asp Arg Leu Leu Ala Ala Ala Ile Glu Asp

260 265 270

atc aag tcc aac acc cgc cgc ctg tcg tgc cgg cag cgc gcc aag atc 1764

Ile Lys Ser Asn Thr Arg Arg Leu Ser Cys Arg Gln Arg Ala Lys Ile

275 280 285

cag cgc ctg gcc aag atg tgg ggc gtc cgg cgc gtc gac gcc acc gag 1812

Gln Arg Leu Ala Lys Met Trp Gly Val Arg Arg Val Asp Ala Thr Glu

290 295 300

gtc ttc cgg agg cgc ggc gac gag gcc gac gag gcc tgg cag cgg ctc 1860

Val Phe Arg Arg Arg Gly Asp Glu Ala Asp Glu Ala Trp Gln Arg Leu

305 310 315

gtc gcc gcg ccg tgc atc gac gcc gtg cgc tcc ttc cta cga acc gac 1908

Val Ala Ala Pro Cys Ile Asp Ala Val Arg Ser Phe Leu Arg Thr Asp

320 325 330 335

gac gcc gcg gcg acc gtc gcc agt gac ctg gcg gtg gac gga gtc gtg 1956

Asp Ala Ala Ala Thr Val Ala Ser Asp Leu Ala Val Asp Gly Val Val

340 345 350

ccc gtg ttc gct ccc gcg ccg gct gcg gcc gta gca gga taa 1998

Pro Val Phe Ala Pro Ala Pro Ala Ala Ala Val Ala Gly *

355 360

cggacgacga agctcctcca tgagcgcgtt gacagctgca ctgcattcac gaatagccat 2058

tatgtataag gacaccatta cttggtgcga tggctgcgat cgtcgtcaag aaggaaaaag 2118

ggagtggtcg agaagcgagg ggaatgacga gcatgattat agtagtatct acgtcggaac 2178

ttgcatggga ctcagtcgca caaagagatt ggcgtgattg cttcgagaat cgtgcgtttg 2238

aaatcctagc tagggagagga ggagagaagc aaacggagaa agtggattta ctatcatata 2298

tatggccgca tatatagcac ttgcatacag ataaatgcgt tacatgtatg ttcgagcgag 2358

gagcatgcat tatttgcacg agttggcacc tgctgtgttt gacttatcac ctgtcctttt 2418

cacccggttt ctgttttatt tgttatatga aagtgatgct agcttattcc tgttttcctt 2478

ttcatttggt tgctacaacg tgcatacatg gttgtgttgt gcgcgcaatc gagtttgtac 2538

aatcgaggtt gtgcatattt acaaggaaat attagttgca aactagcaat attaatgaca 2598

catttttaat atatttatca tatacttcct ccttaaaata taaggcaatc ctttaa 2654

<210>6

<211>364

<212>PRT

<213> corn

<400>6

Met Ser Leu Tyr Leu Ala Pro Thr Ala Ala Ala Ala Thr Thr Thr Thr Thr

1 5 10 15

Thr Thr Leu Pro Arg Gln Leu Leu Pro Ala Pro Ser Ile Asp Leu Arg

20 25 30

Arg Leu Glu Arg Arg Gly Met Ala Leu Pro Leu Pro Pro Ala Pro Ala

35 40 45

Pro Pro Pro Pro Leu Val Ser Ala Asn Asn Arg His Ala Gly Ala Lys

50 55 60

His Lys Ala Val Val Val Met Gly Ala Thr Gly Thr Gly Lys Ser Arg

65 70 75 80

Leu Ala Val Asp Leu Ala Leu Arg Phe Gly Gly Glu Val Ile Asn Ser

85 90 95

Asp Lys Ile Gln Leu His Ala Gly Leu Asp Val Thr Thr Asn Lys Val

100 105 110

Thr Glu Gln Glu Arg Ala Gly Val Pro His His Leu Leu Gly Val Ala

115 120 125

Arg Pro Asp Glu Glu Phe Thr Ala Ala Asp Phe Arg Arg Glu Ala Thr

130 135 140

Arg Ala Ala Arg Ala Ile Thr Ala Arg Gly Arg Leu Pro Ile Val Ala

145 150 155 160

Gly Gly Ser Asn Ser Tyr Val Glu Glu Leu Val Asp Gly Asp Arg Ala

165 170 175

Ala Phe Arg Asp Arg Tyr Asp Cys Cys Phe Leu Trp Val Asp Val Gln

180 185 190

Arg Ala Val Leu His Gly Cys Val Ala Arg Arg Val Asp Glu Met Arg

195 200 205

Ala Arg Gly Leu Val Asp Glu Val Ala Ala Ala Phe Asp Pro Arg Arg

210 215 220

Asn Asp Tyr Ser Arg Gly Leu Trp Arg Ala Ile Gly Ala Pro Glu Leu

225 230 235 240

Asp Ala Tyr Leu Arg Trp Pro Gly Pro Gly Val Asp Gly Asp Ala Glu

245 250 255

Ser Glu Gly Glu Arg Asp Arg Leu Leu Ala Ala Ala Ile Glu Asp Ile

260 265 270

Lys Ser Asn Thr Arg Arg Leu Ser Cys Arg Gln Arg Ala Lys Ile Gln

275 280 285

Arg Leu Ala Lys Met Trp Gly Val Arg Arg Val Asp Ala Thr Glu Val

290 295 300

Phe Arg Arg Arg Gly Asp Glu Ala Asp Glu Ala Trp Gln Arg Leu Val

305 310 315 320

Ala Ala Pro Cys Ile Asp Ala Val Arg Ser Phe Leu Arg Thr Asp Asp

325 330 335

Ala Ala Ala Thr Val Ala Ser Asp Leu Ala Val Asp Gly Val Val Pro

340 345 350

Val Phe Ala Pro Ala Pro Ala Ala Ala Val Ala Gly

355 360

<210>7

<211>1095

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT4 coding sequence

<400>7

atgtcactct acttggcgcc cacggccgcc gctgccacca ccaccaccac caccctccca 60

aggcagctgc taccagcgcc gtccatcgac ctaaggcggc tggaacgacg tggcatggcg 120

ctgcccctgc caccggcgcc ggcgccgccg ccaccgctgg tcagtgccaa caacaggcat 180

gccggagcga agcacaaggc cgtggtggtg atgggcgcca cggggaccgg caagtcgcgg 240

ctggcggtgg acctggcgct ccggttcggc ggcgaggtca tcaactcgga caagatccag 300

ctgcacgccg gcctggacgt gaccacgaac aaggtgaccg agcaggagcg cgccggcgtg 360

ccgcaccacc tgctcggggt ggcgcggccc gacgaggagt tcacggccgc ggacttccgg 420

cgcgaggcga cccgcgccgc gcgcgccatc accgcgcgcg gccgcctgcc catcgtcgcg 480

ggagggtcca actcgtacgt ggaggagctg gtggacggcg accgcgccgc gttccgcgac 540

cgctacgact gctgcttcct ctgggtggac gtgcagcgcg ccgtgctgca cggctgcgtg 600

gcgcggcgcg tcgacgagat gcgcgcccgc ggcctggtgg acgaggtcgc ggcggccttc 660

gacccgcgcc gcaacgacta ctcgcgcggc ctctggcgcg ccattggcgc gcccgagctc 720

gacgcgtacc tgcggtggcc gggaccgggt gtagacggcg acgcggaaag cgagggcgag 780

cgcgaccggc tgctggccgc cgccatcgag gacatcaagt ccaacacccg ccgcctgtcg 840

tgccggcagc gcgccaagat ccagcgcctg gccaagatgt ggggcgtccg gcgcgtcgac 900

gccaccgagg tcttccggag gcgcggcgac gaggccgacg aggcctggca gcggctcgtc 960

gccgcgccgt gcatcgacgc cgtgcgctcc ttcctacgaa ccgacgacgc cgcggcgacc 1020

gtcgccagtg acctggcggt ggacggagtc gtgcccgtgt tcgctcccgc gccggctgcg 1080

gccgtagcag gataa 1095

<210>8

<211>4595

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT5 full-length sequence

<221> CDS

<222>(1952)...(2965)

<400>8

ggaggccggc gccgtggagg cgcgagggtg gtggcccgcc gggttctcgc cggcttacat 60

ggtgtccaag gcggcgctga acgcctactc gcgcgtcctg gcgcggaggc accccgcgct 120

gcgcgtcaac tgcgtgcacc cgggcttcgt caggaccgac atgaccgtca acttcggcat 180

gctcacgccc gaggaaggcg gcagcagggt ggtggccgtc gctctgctcc ccgacggcgg 240

gcccaccggc gcctacttcc aggagcgcca gcaggcgccg ttcgtgtgac gcgctctgcc 300

tctgctctgc ttccttcacc acccgatgct tgatagctat gtatctcttg aagttttcgc 360

ttgataatag ttgatacaca ttcaatcccc gcgcgtaatc tctccgttgc aaaaataatg 420

gaatcctaag ttttggacaa gtaaaaattt ctcaactttt gatcaaccta tcgacgagat 480

gaatcatggg ataaatacat atgttttgcg aagatggaga cacaaaattt ctccatggga 540

atccatcctc gacggagaat tcgacgggtg gatgctgccg agccgtgcat tttcgcactc 600

gtagatgacg cgtctacgca gtacgcaggt gcttcgaact gctgcacgcg ctttgcacag 660

gaacttgcat ttgctgaacc catttcccgt gctcctgttt gtccgctgtt tccatcgtcc 720

gactccgacc ggcggcactc ctctggtctg cctctcgtag caggctagcg gcggctagta 780

ggccccgtgg taccgggccg gaccgattgg attggacgga ggcgggcgcg cacgcaccac 840

acgcccccgg tgtccgggcg tccgtacgcc gtactccctc cccgctgccc gcgtcacgcg 900

tccctccaac caggcgacga aacgactcca ccctccactc actcggccgc cgcacgcagc 960

cctaggccgt agcccaatat gagtgcgcgc cgcggcggct aggcctcccg agtcccgccg 1020

cctccccaac cccgcccagg cgcaggcggg gcggcggcca ccaccagcac ggcacgcacc 1080

acaccaccag tctctgtccg cgatccgatc agtagcgtag cgctagcggc agtcgctctc 1140

gagcgcgggg cgctgttgct gccctgtggt ctgtggacgt ccggcggtcc ggaggccgtg 1200

cagtgcacga tcgtcctgta cgttggaatt ggatcgcctt tgctgctgct gctgcgcgag 1260

tgggtggctc aattcttcga tgcgtttccg acggcccttt tgccctttat tttccctcct 1320

ccttggcagc ttgcggcgga cgacatggtg gtgtcctgtc ctctcgtcgt cctcaccaca 1380

gagaaggcca ccgcagctaa gctagtgcaa accatgcggg gctaagctag taacattatt 1440

ctccgtgcat gcactgtgtc actgcatgat gatcgtcgtt ttcgaaagaa gctagatgac 1500

gaccaagacc agcaggtagt ccgcgcggaa gaaagcgacg agacactcta gtcaactctc 1560

tcttgggcct tggcttgccg tggatataca tggatcggag gtcaactctc ccatggtggc 1620

ttgccgcttg cctgctctgc ctcaatctgt tgccttgttc gtcgtcgtcg tcgtgtcgtg 1680

cgacggcaaa agagaaagcc actcgctgcc ggtacttgca tcgccagtcg ccactcgcca 1740

ccttatctaa accgaatcat gcagtgcggt caaccgcggt cgctgtagcg cgcggccctg 1800

catttggaga cgcccccgcc ctataaatac ccatgctttg ctctgcttct gctcccacac 1860

cacgcagacg cagccaagca agcagccaag gaaggaagct agctagctag ctgatcacaa 1920

agcagggccg attcccgatc cggcagcatc c atg gcg gcc ccc gcg atg gca 1972

                                                                          , 

1 5

gcg ccg ccg cca ccg ccg gcc tgc ttc ccc atg ccc acc agg ctg acg 2020

Ala Pro Pro Pro Pro Pro Ala Cys Phe Pro Met Pro Thr Arg Leu Thr

10 15 20

atg ccg ccg acg tcg atc acg ctc ccg gac ccg ccg ccg ctg tct gtc 2068

Met Pro Pro Thr Ser Ile Thr Leu Pro Asp Pro Pro Pro Leu Ser Val

25 30 35

ggc ggc gcc tgc agg cgc gtg gcg gcc aag cac aag gcc gtg gtg gtg 2116

Gly Gly Ala Cys Arg Arg Val Ala Ala Lys His Lys Ala Val Val Val

40 45 50 55

ctg ggc gcc acg ggc acc ggc aag tcc cgc ctg gcg atc gac ctc gcg 2164

Leu Gly Ala Thr Gly Thr Gly Lys Ser Arg Leu Ala Ile Asp Leu Ala

60 65 70

ctg cgc ttc ggc ggc gag gtc atc aac tcg gac aag atc cag gcg cac 2212

Leu Arg Phe Gly Gly Glu Val Ile Asn Ser Asp Lys Ile Gln Ala His

75 80 85

gcc ggc ctg gac gtg gcc acc aac aag gtg ggc ctc gcc gag cgc ggg 2260

Ala Gly Leu Asp Val Ala Thr Asn Lys Val Gly Leu Ala Glu Arg Gly

90 95 100

cgg gtg ccg cac cac ctg ctg ggc gtg gtg cac ccg gac gcc gag ttc 2308

Arg Val Pro His His Leu Leu Gly Val Val His Pro Asp Ala Glu Phe

105 110 115

acg gcg gcc gac ttc cgc cgc gag gcc tcg cgc gcc gcg gac cgc gcc 2356

Thr Ala Ala Asp Phe Arg Arg Glu Ala Ser Arg Ala Ala Asp Arg Ala

120 125 130 135

gcg gcg cgg ggc cgg gtg ccc gtc atc gcc ggc ggc tcc aac tcg tac 2404

Ala Ala Arg Gly Arg Val Pro Val Ile Ala Gly Gly Ser Asn Ser Tyr

140 145 150

gtg gag gag ctc gtc gag ggg gac cgg cgc gcg ttc cgg gac cgg tac 2452

Val Glu Glu Leu Val Glu Gly Asp Arg Arg Ala Phe Arg Asp Arg Tyr

155 160 165

gag tgc tgc ttc ctc tgg gtg gac gcg cag ctc ccc gtg ctg cac ggc 2500

Glu Cys Cys Phe Leu Trp Val Asp Ala Gln Leu Pro Val Leu His Gly

170 175 180

ttc gtc gcc cgc cgc gtc gac gac atg tgc cgg cgc ggc ctc gtc gac 2548

Phe Val Ala Arg Arg Val Asp Asp Met Cys Arg Arg Gly Leu Val Asp

185 190 195

gag gtg gcg gcc gcg ttc gac ccc cgc cgc acc gac tac tcc agg ggc 2596

Glu Val Ala Ala Ala Phe Asp Pro Arg Arg Thr Asp Tyr Ser Arg Gly

200 205 210 215

atc tgg cgc gcc atc ggc gtg ccg gag ctc gac gcc tac ctc cgg gcg 2644

Ile Trp Arg Ala Ile Gly Val Pro Glu Leu Asp Ala Tyr Leu Arg Ala

220 225 230

cgc ggc cgc ggc cac ggc cac cac cac gac cag atg ctc gcc gcg gcc 2692

Arg Gly Arg Gly His Gly His His His Asp Gln Met Leu Ala Ala Ala

235 240 245

ctc cac gag atc aag gcc aac acg tcc cgc ctc gcc gtg cgc cag cgc 2740

Leu His Glu Ile Lys Ala Asn Thr Ser Arg Leu Ala Val Arg Gln Arg

250 255 260

ggc aag atc cag cgg ctc gag cgc atg tgg cgc gtc cgc cgc gtc gac 2788

Gly Lys Ile Gln Arg Leu Glu Arg Met Trp Arg Val Arg Arg Val Asp

265 270 275

gcc acg gag gtg ttc ctg aag cgc ggc ctc gcc gcc gac gag gcc tgg 2836

Ala Thr Glu Val Phe Leu Lys Arg Gly Leu Ala Ala Asp Glu Ala Trp

280 285 290 295

cag cgg ctg gtc gcc gcg ccc tgc att gac gcc gtc agg tcc ttc ctg 2884

Gln Arg Leu Val Ala Ala Pro Cys Ile Asp Ala Val Arg Ser Phe Leu

300 305 310

ctg gag gac caa caa gag tac agc agc atg ggc acc gcc ggc gcc atg 2932

Leu Glu Asp Gln Gln Glu Tyr Ser Ser Met Gly Thr Ala Gly Ala Met

315 320 325

ttg cct gcc gcc gtc gca gcc gcg gct gtc taa cacaacacaa gtacacaata 2985

Leu Pro Ala Ala Val Ala Ala Ala Ala Val *

330 335

acacaacaca cacggaaccg gaacgctgcc cagagtcctc cacacgtggc cacgcgcgcc 3045

agcctccatt ggcgatcggc gcaatgctct gctgcggtca accgcgcggt gacccggcag 3105

agtagcgcag atcatatagt tgatcctggt caggggaaac tgggaaaagt aatgattttg 3165

tccccatttt ctctgtaata tttttttttg gcacgagata tgatgcgatc gagcgcggga 3225

gcgagttttg agtatggaat aaggccctgt tccaagaatg cgaagtagac gccggcactg 3285

ttggttttgg gggtggtga tgtatttctt attatgagat tagatgtgaa tagtgtctag 3345

tcagtatcgt aatctgaacg tttcatgctc accattgaga tgtgttatca agaatctagc 3405

tggttgcgca cttgcaatag tctagaactc tctctacact cgtttcgatc gcttctgttc 3465

tttttcaata tgtcctttgc attgtgtgat atgaaattgc agcagatatt ggccaaacac 3525

aacaaattgc catgtctctc gtaaatgagc catatttatc cggccggatc atctgcagtt 3585

tcgtccgtgg atgttgctgt agataggccg ctgttgtgtg ggatggtcgt ggtctgctct 3645

atatatcagc ggttagacgt aactggactg ggagttggct gttggcatat gacctgcttc 3705

tgttgtccct ttccatgccc tcgttttcca gctctagcac ttgatgcgag aaacattctc 3765

atgcataatt gagatcagtt agcagatgac ttggaatgcc aattaaggtc aaaccatcgc 3825

accgtcagtg cctgtttgtt gtatttgtat ccgtcctgga gatcaaggtc aactcccagc 3885

gtgctaataa tcggccaaag cctaagcatg gtttcaattc ccagcgtgtg ttatcttctt 3945

gaaactaaca ccgtcaaact ctagaacgta ctagatagtg tgtggccctc aaggtcaacc 4005

aagccacacc ttactagaat cttgtgacgg cagcgagaac gaaccaattg tgcaagtgcc 4065

tttttttctt cttcttcgat ctgtttcgtt tctttgaata gattcttcag gcaacaccac 4125

cagccatgac caaccgcttc gatgccactg atgaatgata atagtcacag atacatagta 4185

cttcacttgc acaacatgat gtgttctaga ggttaaggat ggtgctaaga aagtcaaagc 4245

ttttctatga ggaagaaaaa aaagaagtca aactgcagca tgctctgctc gtttttttaa 4305

atttttttaa tccttttaaa taattttaag atttattttc atcaaaaatt tatttctaaa 4365

catatggtct tttacacttt gcctactagg tgtagtatga ttatttcccc tttctcttac 4425

gtcgtgaaga tgattccgct gcgtcttttg taccatcact ttcgaacttg agggttgttt 4485

cattgtcgta gcacatacac aaggtaccaa gacgtactcg aggaaggatt gatagggaag 4545

taaggaatga gtcataacac cgtacaatat taaggtcatg acatcaagaa 4595

<210>9

<211>337

<212>PRT

<213> corn

<400>9

Met Ala Ala Pro Ala Met Ala Ala Pro Pro Pro Pro Pro Ala Cys Phe

1 5 10 15

Pro Met Pro Thr Arg Leu Thr Met Pro Pro Thr Ser Ile Thr Leu Pro

20 25 30

Asp Pro Pro Pro Leu Ser Val Gly Gly Ala Cys Arg Arg Val Ala Ala

35 40 45

Lys His Lys Ala Val Val Val Leu Gly Ala Thr Gly Thr Gly Lys Ser

50 55 60

Arg Leu Ala Ile Asp Leu Ala Leu Arg Phe Gly Gly Glu Val Ile Asn

65 70 75 80

Ser Asp Lys Ile Gln Ala His Ala Gly Leu Asp Val Ala Thr Asn Lys

85 90 95

Val Gly Leu Ala Glu Arg Gly Arg Val Pro His His Leu Leu Gly Val

100 105 110

Val His Pro Asp Ala Glu Phe Thr Ala Ala Asp Phe Arg Arg Glu Ala

115 120 125

Ser Arg Ala Ala Asp Arg Ala Ala Ala Arg Gly Arg Val Pro Val Ile

130 135 140

Ala Gly Gly Ser Asn Ser Tyr Val Glu Glu Leu Val Glu Gly Asp Arg

145 150 155 160

Arg Ala Phe Arg Asp Arg Tyr Glu Cys Cys Phe Leu Trp Val Asp Ala

165 170 175

Gln Leu Pro Val Leu His Gly Phe Val Ala Arg Arg Val Asp Asp Met

180 185 190

Cys Arg Arg Gly Leu Val Asp Glu Val Ala Ala Ala Phe Asp Pro Arg

195 200 205

Arg Thr Asp Tyr Ser Arg Gly Ile Trp Arg Ala Ile Gly Val Pro Glu

210 215 220

Leu Asp Ala Tyr Leu Arg Ala Arg Gly Arg Gly His Gly His His His His

225 230 235 240

Asp Gln Met Leu Ala Ala Ala Leu His Glu Ile Lys Ala Asn Thr Ser

245 250 255

Arg Leu Ala Val Arg Gln Arg Gly Lys Ile Gln Arg Leu Glu Arg Met

260 265 270

Trp Arg Val Arg Arg Val Asp Ala Thr Glu Val Phe Leu Lys Arg Gly

275 280 285

Leu Ala Ala Asp Glu Ala Trp Gln Arg Leu Val Ala Ala Pro Cys Ile

290 295 300

Asp Ala Val Arg Ser Phe Leu Leu Glu Asp Gln Gln Glu Tyr Ser Ser

305 310 315 320

Met Gly Thr Ala Gly Ala Met Leu Pro Ala Ala Val Ala Ala Ala Ala

325 330 335

Val

<210>10

<211>1014

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT5 coding sequence

<400>10

atggcggccc ccgcgatggc agcgccgccg ccaccgccgg cctgcttccc catgcccacc 60

aggctgacga tgccgccgac gtcgatcacg ctcccggacc cgccgccgct gtctgtcggc 120

ggcgcctgca ggcgcgtggc ggccaagcac aaggccgtgg tggtgctggg cgccacgggc 180

accggcaagt cccgcctggc gatcgacctc gcgctgcgct tcggcggcga ggtcatcaac 240

tcggacaaga tccaggcgca cgccggcctg gacgtggcca ccaacaaggt gggcctcgcc 300

gagcgcgggc gggtgccgca ccacctgctg ggcgtggtgc acccggacgc cgagttcacg 360

gcggccgact tccgccgcga ggcctcgcgc gccgcggacc gcgccgcggc gcggggccgg 420

gtgcccgtca tcgccggcgg ctccaactcg tacgtggagg agctcgtcga gggggaccgg 480

cgcgcgttcc gggaccggta cgagtgctgc ttcctctggg tggacgcgca gctccccgtg 540

ctgcacggct tcgtcgcccg ccgcgtcgac gacatgtgcc ggcgcggcct cgtcgacgag 600

gtggcggccg cgttcgaccc ccgccgcacc gactactcca ggggcatctg gcgcgccatc 660

ggcgtgccgg agctcgacgc ctacctccgg gcgcgcggcc gcggccacgg ccaccaccac 720

gaccagatgc tcgccgcggc cctccacgag atcaaggcca acacgtcccg cctcgccgtg 780

cgccagcgcg gcaagatcca gcggctcgag cgcatgtggc gcgtccgccg cgtcgacgcc 840

acggaggtgt tcctgaagcg cggcctcgcc gccgacgagg cctggcagcg gctggtcgcc 900

gcgccctgca ttgacgccgt caggtccttc ctgctggagg accaacaaga gtacagcagc 960

atgggcaccg ccggcgccat gttgcctgcc gccgtcgcag ccgcggctgt ctaa 1014

<210>11

<211>1955

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT6 full-length sequence

<221> CDS

<222>(659)...(1675)

<400>11

aataacattt tagtaatttg tttttctttt atttatatgg accagcagct agtgtatgtg 60

aggggctgta aaaatggttc tttacatcct acatgtaaag aattttttcc cctcccatat 120

gcatacaagc tctttgaatg aaagaactgc agtggtgtgg gtctcaggca aacaatccac 180

gtcggaggag gagtgttacg gcgttgcatt tcaccacggt ttactggtga gtgatttttt 240

ttttctttaa ggcctccttt agaacacatg gattttatag aaatgatata tgaattttac 300

agaaaatagt tcaaaaccat agaaaaaatg caggattcac gaaataatcg tttgttccaa 360

aggatgcgtt agacttcggc aaatcccatt cgccgcatcc aatccatcca ctacggagtg 420

ttcagccaaa gctttaaact agtcgtcatt cgtcaagaac acaaccacca gcggacaacc 480

cgaccagtga aaccctctgt cccgccctat aaatacccat gctttcttct gctcctacac 540

cacgcaccca agcaagccaa gcgacgacca gcagcaagca gccaaggaag ctagctagct 600

agctgaccga ccagctgtga tcgcaagcag ggccgacccc gaccggcagc gtatatac 658

atg aca ctg tcc atg acg gcc ccc gcg atg gca gca gcg cca ccg gcc 706

Met Thr Leu Ser Met Thr Ala Pro Ala Met Ala Ala Ala Pro Pro Ala

1 5 10 15

tgc tcc ccc atg gcg gcc agg ctg acg atg ccg ccg ctg ccg gac gcc 754

Cys Ser Pro Met Ala Ala Arg Leu Thr Met Pro Pro Leu Pro Asp Ala

20 25 30

gcg gcg ccg ctg tcg gtc gtg ggc tgc agg cgc atg gcg gcc aag cac 802

Ala Ala Pro Leu Ser Val Val Gly Cys Arg Arg Met Ala Ala Lys His

35 40 45

aag gcc gtc gtg gtg ctg ggc gcc acg ggc acc ggc aag tcc cgc ctg 850

Lys Ala Val Val Val Leu Gly Ala Thr Gly Thr Gly Lys Ser Arg Leu

50 55 60

gcg atc gac ctc gct ctg cgc ttc ggc ggc gag gtc atc aac tcc gac 898

Ala Ile Asp Leu Ala Leu Arg Phe Gly Gly Glu Val Ile Asn Ser Asp

65 70 75 80

aag atc cag gcg tac gcc ggc ctg gac gtg gcc acc aac aag gtg ggg 946

Lys Ile Gln Ala Tyr Ala Gly Leu Asp Val Ala Thr Asn Lys Val Gly

85 90 95

ccc gcc gag cgc gcg gcg gtg ccg cac cac ctg ctg ggc gtc gtg cac 994

Pro Ala Glu Arg Ala Ala Val Pro His His Leu Leu Gly Val Val His

100 105 110

ccg gac gcc gag ttc acg gcg gcg gac ttc cgg cgc gag gcc gcg ggc 1042

Pro Asp Ala Glu Phe Thr Ala Ala Asp Phe Arg Arg Glu Ala Ala Gly

115 120 125

gcc gcg gcc cgc gtc gcg tcg cgg ggc cgc gtg ccc atc atc gcg ggc 1090

Ala Ala Ala Arg Val Ala Ser Arg Gly Arg Val Pro Ile Ile Ala Gly

130 135 140

ggc tcc aac tcg tac gtg gag gag ctc gtc gag ggg gac cgc cgc gcg 1138

Gly Ser Asn Ser Tyr Val Glu Glu Leu Val Glu Gly Asp Arg Arg Ala

145 150 155 160

ttc cgg gag agg tac gac tgc tgc ttc ctg tgg gtg gac gcg cgg ctc 1186

Phe Arg Glu Arg Tyr Asp Cys Cys Phe Leu Trp Val Asp Ala Arg Leu

165 170 175

ccc gtg ctg cac ggc ttc gtg gcc cgc cgc gtg gac gag atg tgc cgg 1234

Pro Val Leu His Gly Phe Val Ala Arg Arg Val Asp Glu Met Cys Arg

180 185 190

cgc ggg ctc gtg gac gag gtg gcg gcc gcg ttc gac ccg cgc cgc acc 1282

Arg Gly Leu Val Asp Glu Val Ala Ala Ala Phe Asp Pro Arg Arg Thr

195 200 205

gac tac tcg agg ggc atc tgg cgc gcc atc ggc gtg ccg gag atg gac 1330

Asp Tyr Ser Arg Gly Ile Trp Arg Ala Ile Gly Val Pro Glu Met Asp

210 215 220

gcc tac ctc cgc gcg ggc ggc cac ggc gac ggc gac ggc gac gag cag 1378

Ala Tyr Leu Arg Ala Gly Gly His Gly Asp Gly Asp Gly Asp Glu Gln

225 230 235 240

gag cag cgc gcg cgc atg ctc gcc gcg gcg ctc gac gag atc aag gtc 1426

Glu Gln Arg Ala Arg Met Leu Ala Ala Ala Leu Asp Glu Ile Lys Val

245 250 255

aac acg tcc cgg ctc gcc ctg cgt cag cgc ggc aag atc cag cgg ctg 1474

Asn Thr Ser Arg Leu Ala Leu Arg Gln Arg Gly Lys Ile Gln Arg Leu

260 265 270

gca cgc atg tgg cgc gtc cgc cgc gtc gac gcc acg gag gtg ttc ctg 1522

Ala Arg Met Trp Arg Val Arg Arg Val Asp Ala Thr Glu Val Phe Leu

275 280 285

aag cgc ggc cac gcc gcc gac gag gcc tgg cag cgg ctg gtc gcc gcg 1570

Lys Arg Gly His Ala Ala Asp Glu Ala Trp Gln Arg Leu Val Ala Ala

290 295 300

ccg tgc att gac gcc gtc agg tcc ttc ctg ctg gag gag caa gag tac 1618

Pro Cys Ile Asp Ala Val Arg Ser Phe Leu Leu Glu Glu Gln Glu Tyr

305 310 315 320

agc agc agc atg gtc acc gcc tcc atg ttt gcc tcc acg gcg gcc gcg 1666

Ser Ser Ser Met Val Thr Ala Ser Met Phe Ala Ser Thr Ala Ala Ala

325 330 335

gct gtc tag cctcccccca gtggctcacc agctcagcga atggaagaat 1715

Ala Val *

gaatggccag cttggctgtg ctgcatgctc tgctgcggtc aaccgcgcgc tgaccggcag 1775

agtggcgctg atgatatagt tgaccctgtc acaagagcag ggaaagtaat tattttgtcc 1835

ccattttctc tgtatttttg tacgagatga tgcgatcgag cgcgagagcg agttttgagt 1895

accctgtccc aagagcactg ttgatttggg gtgagactgt gttcagcggt tacccctaaa 1955

<210>12

<211>338

<212>PRT

<213> corn

<400>12

Met Thr Leu Ser Met Thr Ala Pro Ala Met Ala Ala Ala Pro Pro Ala

1 5 10 15

Cys Ser Pro Met Ala Ala Arg Leu Thr Met Pro Pro Leu Pro Asp Ala

20 25 30

Ala Ala Pro Leu Ser Val Val Gly Cys Arg Arg Met Ala Ala Lys His

35 40 45

Lys Ala Val Val Val Leu Gly Ala Thr Gly Thr Gly Lys Ser Arg Leu

50 55 60

Ala Ile Asp Leu Ala Leu Arg Phe Gly Gly Glu Val Ile Asn Ser Asp

65 70 75 80

Lys Ile Gln Ala Tyr Ala Gly Leu Asp Val Ala Thr Asn Lys Val Gly

85 90 95

Pro Ala Glu Arg Ala Ala Val Pro His His Leu Leu Gly Val Val His

100 105 110

Pro Asp Ala Glu Phe Thr Ala Ala Asp Phe Arg Arg Glu Ala Ala Gly

115 120 125

Ala Ala Ala Arg Val Ala Ser Arg Gly Arg Val Pro Ile Ile Ala Gly

130 135 140

Gly Ser Asn Ser Tyr Val Glu Glu Leu Val Glu Gly Asp Arg Arg Ala

145 150 155 160

Phe Arg Glu Arg Tyr Asp Cys Cys Phe Leu Trp Val Asp Ala Arg Leu

165 170 175

Pro Val Leu His Gly Phe Val Ala Arg Arg Val Asp Glu Met Cys Arg

180 185 190

Arg Gly Leu Val Asp Glu Val Ala Ala Ala Phe Asp Pro Arg Arg Thr

195 200 205

Asp Tyr Ser Arg Gly Ile Trp Arg Ala Ile Gly Val Pro Glu Met Asp

210 215 220

Ala Tyr Leu Arg Ala Gly Gly His Gly Asp Gly Asp Gly Asp Glu Gln

225 230 235 240

Glu Gln Arg Ala Arg Met Leu Ala Ala Ala Leu Asp Glu Ile Lys Val

245 250 255

Asn Thr Ser Arg Leu Ala Leu Arg Gln Arg Gly Lys Ile Gln Arg Leu

260 265 270

Ala Arg Met Trp Arg Val Arg Arg Val Asp Ala Thr Glu Val Phe Leu

275 280 285

Lys Arg Gly His Ala Ala Asp Glu Ala Trp Gln Arg Leu Val Ala Ala

290 295 300

Pro Cys Ile Asp Ala Val Arg Ser Phe Leu Leu Glu Glu Gln Glu Tyr

305 310 315 320

Ser Ser Ser Met Val Thr Ala Ser Met Phe Ala Ser Thr Ala Ala Ala

325 330 335

Ala Val

<210>13

<211>1017

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT6 coding sequence

<400>13

atgacactgt ccatgacggc ccccgcgatg gcagcagcgc caccggcctg ctcccccatg 60

gcggccaggc tgacgatgcc gccgctgccg gacgccgcgg cgccgctgtc ggtcgtgggc 120

tgcaggcgca tggcggccaa gcacaaggcc gtcgtggtgc tgggcgccac gggcaccggc 180

aagtcccgcc tggcgatcga cctcgctctg cgcttcggcg gcgaggtcat caactccgac 240

aagatccagg cgtacgccgg cctggacgtg gccaccaaca aggtggggcc cgccgagcgc 300

gcggcggtgc cgcaccacct gctgggcgtc gtgcacccgg acgccgagtt cacggcggcg 360

gacttccggc gcgaggccgc gggcgccgcg gcccgcgtcg cgtcgcgggg ccgcgtgccc 420

atcatcgcgg gcggctccaa ctcgtacgtg gaggagctcg tcgaggggga ccgccgcgcg 480

ttccgggaga ggtacgactg ctgcttcctg tgggtggacg cgcggctccc cgtgctgcac 540

ggcttcgtgg cccgccgcgt ggacgagatg tgccggcgcg ggctcgtgga cgaggtggcg 600

gccgcgttcg acccgcgccg caccgactac tcgaggggca tctggcgcgc catcggcgtg 660

ccggagatgg acgcctacct ccgcgcgggc ggccacggcg acggcgacgg cgacgagcag 720

gagcagcgcg cgcgcatgct cgccgcggcg ctcgacgaga tcaaggtcaa cacgtcccgg 780

ctcgccctgc gtcagcgcgg caagatccag cggctggcac gcatgtggcg cgtccgccgc 840

gtcgacgcca cggaggtgtt cctgaagcgc ggccacgccg ccgacgaggc ctggcagcgg 900

ctggtcgccg cgccgtgcat tgacgccgtc aggtccttcc tgctggagga gcaagagtac 960

agcagcagca tggtcaccgc ctccatgttt gcctccacgg cggccgcggc tgtctag 1017

<210>14

<211>1652

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT7 full-length sequence

<221> CDS

<222>(298)...(1356)

<400>14

tcaaagactg aaaagtaatt taattgaccc ccggtcaggt caggtcaggt caggtcatgt 60

caggcacaag gcgtgtgtta gggtttcctt gtcgcgtact aacgaagaca tacatacgaa 120

ctgacgggaa aacaccagtc aagtctgtgg ctgacttcgt gccgtgcgct cttgaacccg 180

agcactataa aaacggagac ctatgcacgc ttttcattca cccatataca cagccgctac 240

actactacaa gctagcttgt acgtggtaac actgtgaaag acagtcgtac actcacg atg 300

Met

1

gcg gga gtt aac ggt gcc acg gcg agc gga ggc gac aac aag gcc aag 348

Ala Gly Val Asn Gly Ala Thr Ala Ser Gly Gly Asp Asn Lys Ala Lys

5 10 15

gtg gtg ctg gtg atg ggc gcc acg gcc acc ggc aag tcc aag ctg gcc 396

Val Val Leu Val Met Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu Ala

20 25 30

atc gac ctc gcg ctg cgc ttc ggc gga gag gtc gtc aac tcc gac aag 444

Ile Asp Leu Ala Leu Arg Phe Gly Gly Glu Val Val Asn Ser Asp Lys

35 40 45

atc cag gtg cac gac ggc ctc gac gtg gtc acc aac aag gtc acc gcc 492

Ile Gln Val His Asp Gly Leu Asp Val Val Thr Asn Lys Val Thr Ala

50 55 60 65

gcg gag cgc cag ggc gtg cca cac cac ctt atc gac ggg gtg gcg ccc 540

Ala Glu Arg Gln Gly Val Pro His His Leu Ile Asp Gly Val Ala Pro

70 75 80

gac gcc gac tac acc acc gcc gac ttc tgc agg gac gcc gtg cgc gct 588

Asp Ala Asp Tyr Thr Thr Ala Asp Phe Cys Arg Asp Ala Val Arg Ala

85 90 95

gtg gag tcc att ctc gag agg ggc cgc gtc ccg atc atc gcc ggg ggc 636

Val Glu Ser Ile Leu Glu Arg Gly Arg Val Pro Ile Ile Ala Gly Gly

100 105 110

tcc aac aga tac ctg gag gcg ctg ctg gac ggg gaa cct cct gca ggc 684

Ser Asn Arg Tyr Leu Glu Ala Leu Leu Asp Gly Glu Pro Pro Ala Gly

115 120 125

ttc cgc ggc cgc tac gaa tgc tgc ttc ctc tgg gtc gac agc gac ctg 732

Phe Arg Gly Arg Tyr Glu Cys Cys Phe Leu Trp Val Asp Ser Asp Leu

130 135 140 145

gcg gtg ytg gac cgc tac ata ggg agc cgc gtg gac tgc atg ctg gag 780

Ala Val Xaa Asp Arg Tyr Ile Gly Ser Arg Val Asp Cys Met Leu Glu

150 155 160

cag ggg ctc gtc cgc gag gtg cgg gcc ttc ttt cgg cac gac gac gcc 828

Gln Gly Leu Val Arg Glu Val Arg Ala Phe Phe Arg His Asp Asp Ala

165 170 175

gac tac tcc agg ggt atc cgg agg gcc atc ggc gtg ccg gag atg gac 876

Asp Tyr Ser Arg Gly Ile Arg Arg Ala Ile Gly Val Pro Glu Met Asp

180 185 190

atg tac ttc cgg atg gag gcc gca ggg gct ctc gac ggc gac gat gat 924

Met Tyr Phe Arg Met Glu Ala Ala Gly Ala Leu Asp Gly Asp Asp Asp

195 200 205

gat cag ctg cga gtg cgg ctc ctc gcc gcg gcc gtt aac gag atc aag 972

Asp Gln Leu Arg Val Arg Leu Leu Ala Ala Ala Val Asn Glu Ile Lys

210 215 220 225

gcc aac acg tgc ggc ctg gcg cgc cgc cag ctg cag aag atc cac cgg 1020

Ala Asn Thr Cys Gly Leu Ala Arg Arg Gln Leu Gln Lys Ile His Arg

230 235 240

ctc cac ggt ctc caa ggc tgg agc gac atc cac cgc ctc gac gtc acg 1068

Leu His Gly Leu Gln Gly Trp Ser Asp Ile His Arg Leu Asp Val Thr

245 250 255

gag gtg ctt cag ctc aag gtc ggg aac gcc ggg aac cca aag gca cag 1116

Glu Val Leu Gln Leu Lys Val Gly Asn Ala Gly Asn Pro Lys Ala Gln

260 265 270

cgc gac gcg tgg gag acg gac gtc gtc agc cct gcg gcg agg atc gtg 1164

Arg Asp Ala Trp Glu Thr Asp Val Val Ser Pro Ala Ala Arg Ile Val

275 280 285

gga atg ttt ctg gct gtt gag gga gct agg gac aag gac aag gac cgt 1212

Gly Met Phe Leu Ala Val Glu Gly Ala Arg Asp Lys Asp Lys Asp Arg

290 295 300 305

ttc ttg ttg acg acg ccc aaa gaa gtg gcc gtg cca ggc att tgc acg 1260

Phe Leu Leu Thr Thr Pro Lys Glu Val Ala Val Pro Gly Ile Cys Thr

310 315 320

gcc acg gca gat tgg ttc ggc cag cag ctg gac atg acg gtc atg tct 1308

Ala Thr Ala Asp Trp Phe Gly Gln Gln Leu Asp Met Thr Val Met Ser

325 330 335

cca agc aaa ggg ttt gct gga ttg ggc tcg gcg gcc gcc gcg gtt taa 1356

Pro Ser Lys Gly Phe Ala Gly Leu Gly Ser Ala Ala Ala Ala Val *

340 345 350

gctgtgatga cgatgaactc atctgctgat gatcaatcac tgaacggact ccaaatgcat 1416

gtacattcat ttatttcatg tagggaataa gttgttgtaa tgtattgctc ccccactaat 1476

aattaattta aggccaggta taggagaaat tgtaggggag gctgaacgac atagctcata 1536

ctttggaacg ttgtaatcta gccgatcaat ttcacgaaac catatttttt gaaacataat 1596

ctctattcag cattatgcca cgccattatac acaagacctt gtatatatat aaaaag 1652

<210>15

<211>352

<212>PRT

<213> corn

<220>

<221> variant

<222>148

<223> Xaa = any amino acid

<400>15

Met Ala Gly Val Asn Gly Ala Thr Ala Ser Gly Gly Asp Asn Lys Ala

1 5 10 15

Lys Val Val Leu Val Met Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu

20 25 30

Ala Ile Asp Leu Ala Leu Arg Phe Gly Gly Glu Val Val Asn Ser Asp

35 40 45

Lys Ile Gln Val His Asp Gly Leu Asp Val Val Thr Asn Lys Val Thr

50 55 60

Ala Ala Glu Arg Gln Gly Val Pro His His Leu Ile Asp Gly Val Ala

65 70 75 80

Pro Asp Ala Asp Tyr Thr Thr Ala Asp Phe Cys Arg Asp Ala Val Arg

85 90 95

Ala Val Glu Ser Ile Leu Glu Arg Gly Arg Val Pro Ile Ile Ala Gly

100 105 110

Gly Ser Asn Arg Tyr Leu Glu Ala Leu Leu Asp Gly Glu Pro Pro Ala

115 120 125

Gly Phe Arg Gly Arg Tyr Glu Cys Cys Phe Leu Trp Val Asp Ser Asp

130 135 140

Leu Ala Val Xaa Asp Arg Tyr Ile Gly Ser Arg Val Asp Cys Met Leu

145 150 155 160

Glu Gln Gly Leu Val Arg Glu Val Arg Ala Phe Phe Arg His Asp Asp

165 170 175

Ala Asp Tyr Ser Arg Gly Ile Arg Arg Ala Ile Gly Val Pro Glu Met

180 185 190

Asp Met Tyr Phe Arg Met Glu Ala Ala Gly Ala Leu Asp Gly Asp Asp

195 200 205

Asp Asp Gln Leu Arg Val Arg Leu Leu Ala Ala Ala Val Asn Glu Ile

210 215 220

Lys Ala Asn Thr Cys Gly Leu Ala Arg Arg Gln Leu Gln Lys Ile His

225 230 235 240

Arg Leu His Gly Leu Gln Gly Trp Ser Asp Ile His Arg Leu Asp Val

245 250 255

Thr Glu Val Leu Gln Leu Lys Val Gly Asn Ala Gly Asn Pro Lys Ala

260 265 270

Gln Arg Asp Ala Trp Glu Thr Asp Val Val Ser Pro Ala Ala Arg Ile

275 280 285

Val Gly Met Phe Leu Ala Val Glu Gly Ala Arg Asp Lys Asp Lys Asp

290 295 300

Arg Phe Leu Leu Thr Thr Pro Lys Glu Val Ala Val Pro Gly Ile Cys

305 310 315 320

Thr Ala Thr Ala Asp Trp Phe Gly Gln Gln Leu Asp Met Thr Val Met

325 330 335

Ser Pro Ser Lys Gly Phe Ala Gly Leu Gly Ser Ala Ala Ala Ala Val

340 345 350

<210>16

<211>1059

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT7 coding sequence

<400>16

atggcgggag ttaacggtgc cacggcgagc ggaggcgaca acaaggccaa ggtggtgctg 60

gtgatgggcg ccacggccac cggcaagtcc aagctggcca tcgacctcgc gctgcgcttc 120

ggcggagagg tcgtcaactc cgacaagatc caggtgcacg acggcctcga cgtggtcacc 180

aacaaggtca ccgccgcgga gcgccagggc gtgccacacc accttatcga cggggtggcg 240

cccgacgccg actacaccac cgccgacttc tgcagggacg ccgtgcgcgc tgtggagtcc 300

attctcgaga ggggccgcgt cccgatcatc gccgggggct ccaacagata cctggaggcg 360

ctgctggacg gggaacctcc tgcaggcttc cgcggccgct acgaatgctg cttcctctgg 420

gtcgacagcg acctggcggt gytggaccgc tacataggga gccgcgtgga ctgcatgctg 480

gagcaggggc tcgtccgcga ggtgcgggcc ttctttcggc acgacgacgc cgactactcc 540

aggggtatcc ggagggccat cggcgtgccg gagatggaca tgtacttccg gatggaggcc 600

gcaggggctc tcgacggcga cgatgatgat cagctgcgag tgcggctcct cgccgcggcc 660

gttaacgaga tcaaggccaa cacgtgcggc ctggcgcgcc gccagctgca gaagatccac 720

cggctccacg gtctccaagg ctggagcgac atccaccgcc tcgacgtcac ggaggtgctt 780

cagctcaagg tcgggaacgc cgggaaccca aaggcacagc gcgacgcgtg ggagacggac 840

gtcgtcagcc ctgcggcgag gatcgtggga atgtttctgg ctgttgaggg agctagggac 900

aaggacaagg accgtttctt gttgacgacg cccaaagaag tggccgtgcc aggcatttgc 960

acggccacgg cagattggtt cggccagcag ctggacatga cggtcatgtc tccaagcaaa 1020

gggtttgctg gattgggctc ggcggccgcc gcggtttaa 1059

<210>17

<211>3419

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT8 full-length sequence

<221> CDS

<222>(1897)...(3063)

<400>17

gtcttagtgc atcgattcta agagtgaagc aggtagaata ctcacctatt ctctgtaaga 60

tatatgagtg agtttaaata cctctctaca gaccaaattc tagagttttt tttttccaaa 120

ccgatactga tcggtctcca gtggttggat aaataccgac cttattattc ggtcagcagg 180

cgaagcgacc gcagtgttag cgatcgacga taaaaccatg gtggtcagca ctgatcgcag 240

gcgaggccac gtcgtcgttc cgtaacgctc gtgctgaaac cagcccgagt aaactgtgag 300

ccgcttcttt ttcgagtacg atccgggacc agacgttaca gaaaaaaaaa aggatgagca 360

tgaagctgct gccgaggctg agcatcaagg ctgcctttcc gttcattggc tgggtgcggt 420

tatgggtgca gtactgcagt gctgcgcagt cgagtcgtgc tcgtctccat cctactcacc 480

atgtgcagcc gactttcata tagaaccgga ggctgacaga cagacagaca gacagagccg 540

ctacgctact ggatcaccgc cggccagggg aacgagaggc atggccgcat catcaccagc 600

catgggttag tagtcaccca aattcttcct cgcatcgcac gcgccgcgcg gtcaagccac 660

gacacgagac ggaggattac gaaaaggaac ggaacggaac cgcccgtcgc gtcgcacgcc 720

gggaccggga tgggcgcgga cgcgagcccg gaggcaggcc gaaaggtcgc cggccggctg 780

gccggacgga cggaagggga ggcggggaga aacgaagcct gtcgtgtcgt gtcgtgtcgt 840

gcgaggcagc cagcgccgca ggtcgcagtc tctggagtct ggacccggcc gaaaaggcgt 900

gccgggacca ggaccagaac ccggacccgg atccaacaga agatgtacta ttggcaggag 960

cagtcggaag cggacgcgcg cgcacccgac acgatggggg cgccagtgcc accaactcag 1020

cagccagtgg ccggccgggc ccaaagcggc tggagcttgc cgtgccggct gcctgccgaa 1080

tccaagcgaa gcgcaccggc gcggctaccc gcacccgctc gcggctgccc tgccctccca 1140

ctagtcccgg ctctgctgct cgcctcggaa tgttctcgcc gcctgcgggg acgacacgcg 1200

acgcttatta taaccacgcc tctgtccggc ggtcgccctg ccaccgcccg gcacccggca 1260

cccgccacct ggttttgttg gcgtccctcg cgctgccact gcgagctggg tggggatcgg 1320

ggccccggtc catccggaca ggaagccccg catggccgcg accgccatca tgggccgacg 1380

tacagcgtcc gcttttgttt aaggtccccc gcgcatcgca tcgatcgtgt cgtgtcgtgg 1440

caattgcttt cggagtgtcg agcgccactc ctgcaaagcc tgtgctgtgc tgctgccttg 1500

tattgtatat atacacacgc acacacaacg agtcgagccg agcaatactg gcacggcgcg 1560

ttgccccctg ccagacagca cggccaacgc caccccacca gtccaacagc aggcggtggc 1620

cgaggagggag ggaatagccg aatagggatt tttgaggttt tcggcggcag aaatacgagc 1680

gaaaactcaa aacaggcgcg cgactgaaga gagagcgcta gctggaagag acgggcgagc 1740

cgatctggcg tggcagccgt tccctgcccc gtccccgcat aaatcccaac aaggacgctg 1800

gcgctgcgtg tatctccctc gcaagagaca aaaaaaaata tcacacacac ggcgcggcgg 1860

tcatagtgcg gctgattcgg atcgggcact agctac atg acc acc ctc ctc gcc 1914

                        

1 5

aat agg atc act acg ctc gtg cgc gcc cct cct cct ccc atg gcc gcc 1962

Asn Arg Ile Thr Thr Leu Val Arg Ala Pro Pro Pro Pro Met Ala Ala

10 15 20

gcc gcc gtc gcg gga gcg cgg agg cca ttg cac cgg acc ttg gcg cac 2010

Ala Ala Val Ala Gly Ala Arg Arg Pro Leu His Arg Thr Leu Ala His

25 30 35

ccg cca ccg ccc gag gag gac gag cat cag cag cag cgc gcg tgc cgc 2058

Pro Pro Pro Pro Glu Glu Asp Glu His Gln Gln Gln Arg Ala Cys Arg

40 45 50

agc agg gga tcc tcg tcc tcc tgc tcg gct tcc tcg tca tcg acg ccc 2106

Ser Arg Gly Ser Ser Ser Ser Cys Ser Ala Ser Ser Ser Ser Thr Pro

55 60 65 70

gcc cgg ccc cgg ggc acg ggg atg gtg gtg atc gtc ggc gcc acg ggc 2154

Ala Arg Pro Arg Gly Thr Gly Met Val Val Ile Val Gly Ala Thr Gly

75 80 85

acc ggg aag acc aag ctg tcc atc gac gcc gcg gag gcg gtc ggc ggg 2202

Thr Gly Lys Thr Lys Leu Ser Ile Asp Ala Ala Glu Ala Val Gly Gly

90 95 100

gag gtg gtg aac gcg gat aag atc cag ctc tac gcc ggg ctg gac gtg 2250

Glu Val Val Asn Ala Asp Lys Ile Gln Leu Tyr Ala Gly Leu Asp Val

105 110 115

acc acg aac aag gtg gcc ccc gcg gac cgc cgc ggc gtg ccg cac cac 2298

Thr Thr Asn Lys Val Ala Pro Ala Asp Arg Arg Gly Val Pro His His

120 125 130

ctc ctc ggc gcc atc cgc ccc gag gcc ggc gag ctc ccg ccc tcc acg 2346

Leu Leu Gly Ala Ile Arg Pro Glu Ala Gly Glu Leu Pro Pro Ser Thr

135 140 145 150

ttc cgc tcc ctc gcc gcc gcc acg gcc gcc tcg atc gcc gcg cgc ggc 2394

Phe Arg Ser Leu Ala Ala Ala Thr Ala Ala Ser Ile Ala Ala Arg Gly

155 160 165

cgc ctg ccg gtc gtc gcg ggc ggc tcc aac tcc ctc atc cac gcg ctc 2442

Arg Leu Pro Val Val Ala Gly Gly Ser Asn Ser Leu Ile His Ala Leu

170 175 180

ctc gcc gac cgc ctc gac gcc ggc gcc gcc gac ccc ttc tcc gct cca 2490

Leu Ala Asp Arg Leu Asp Ala Gly Ala Ala Asp Pro Phe Ser Ala Pro

185 190 195

ccg cag ccg gcg ccg ccg cgg tgg ggc cgc cgg ccc gcg ctc cga tcc 2538

Pro Gln Pro Ala Pro Pro Arg Trp Gly Arg Arg Pro Ala Leu Arg Ser

200 205 210

ccg tgc tgt ctc ctc tgg gtc cac gtc gac gcc gcg ctc ctc gcg gag 2586

Pro Cys Cys Leu Leu Trp Val His Val Asp Ala Ala Leu Leu Ala Glu

215 220 225 230

tac ctg gac cgg cgc gtg gac gac atg gtg cgc ggc ggc atg gtg gag 2634

Tyr Leu Asp Arg Arg Val Asp Asp Met Val Arg Gly Gly Met Val Glu

235 240 245

gag ctg cgg gag tac ttc gcc gcg acc acc gcc gcc gag cgc gcc gcg 2682

Glu Leu Arg Glu Tyr Phe Ala Ala Thr Thr Ala Ala Glu Arg Ala Ala

250 255 260

cac gcc gcg ggg ctg ggc agg gcc atc ggc gtg ccc gag ctg ggc gcc 2730

His Ala Ala Gly Leu Gly Arg Ala Ile Gly Val Pro Glu Leu Gly Ala

265 270 275

tgc ttc gcg ggg cgc gcc agc ttc cgc gcc gcg atc gac gac atc aag 2778

Cys Phe Ala Gly Arg Ala Ser Phe Arg Ala Ala Ile Asp AspIle Lys

280 285 290

gcc aac acg cgg gac ctg gcg gcc gcg cag gtg cgc aag atc cga cgc 2826

Ala Asn Thr Arg Asp Leu Ala Ala Ala Gln Val Arg Lys Ile Arg Arg

295 300 305 310

atg gcc gat gcc tgg ggc tgg ccc atc cag cgg ctc gac gcg tcg gcc 2874

Met Ala Asp Ala Trp Gly Trp Pro Ile Gln Arg Leu Asp Ala Ser Ala

315 320 325

aca gtc cgc gcg cgc ctc cgc ggc gcg ggg ccc gac gcg gag tcg gcg 2922

Thr Val Arg Ala Arg Leu Arg Gly Ala Gly Pro Asp Ala Glu Ser Ala

330 335 340

tgc tgg gag cgc gac gtg cgc gcg ccc ggg ctc gcc gcc atc cgg agc 2970

Cys Trp Glu Arg Asp Val Arg Ala Pro Gly Leu Ala Ala Ile Arg Ser

345 350 355

ttc ctt cta gag ctg gac ggc ggc agc gtc gtc gac ggc gct gtg gtg 3018

Phe Leu Leu Glu Leu Asp Gly Gly Ser Val Val Asp Gly Ala Val Val

360 365 370

gag gag gtg gag ccg cgg gtg cga tgc tgc gac gtg gtg ggg tga 3063

Glu Glu Val Glu Pro Arg Val Arg Cys Cys Asp Val Val Gly *

375 380 385

gcgagctcgg tcctcagctg ctgtcacttt ccgggcggag ttattcgcga tatacgccgc 3123

gaaaaggctg cggggggctt ttggactcga gggtttaggc cgccgatttt gcagggtccc 3183

ggcggccgct gaccggtggg gtccggcgaa gggcacagcg agtgagtgag tgacacaggg 3243

acatgagaat gagagaagga gacagaggga gaaaagaaaa tgcttcatgt ttagtgttta 3303

ctacaaatca ttactattag ttaccattag tgtaggcaga gataagcatt gatgaaggga 3363

gagggaggag actgtgaatt cgaggggtat cttttcttct ttttgctttt ggttcg 3419

<210>18

<211>388

<212>PRT

<213> corn

<400>18

Met Thr Thr Leu Leu Ala Asn Arg Ile Thr Thr Leu Val Arg Ala Pro

1 5 10 15

Pro Pro Pro Met Ala Ala Ala Ala Val Ala Gly Ala Arg Arg Pro Leu

20 25 30

His Arg Thr Leu Ala His Pro Pro Pro Pro Glu Glu Asp Glu His Gln

35 40 45

Gln Gln Arg Ala Cys Arg Ser Arg Gly Ser Ser Ser Ser Cys Ser Ala

50 55 60

Ser Ser Ser Ser Ser Thr Pro Ala Arg Pro Arg Gly Thr Gly Met Val Val

65 70 75 80

Ile Val Gly Ala Thr Gly Thr Gly Lys Thr Lys Leu Ser Ile Asp Ala

85 90 95

Ala Glu Ala Val Gly Gly Glu Val Val Asn Ala Asp Lys Ile Gln Leu

100 105 110

Tyr Ala Gly Leu Asp Val Thr Thr Asn Lys Val Ala Pro Ala Asp Arg

115 120 125

Arg Gly Val Pro His His Leu Leu Gly Ala Ile Arg Pro Glu Ala Gly

130 135 140

Glu Leu Pro Pro Ser Thr Phe Arg Ser Leu Ala Ala Ala Thr Ala Ala

145 150 155 160

Ser Ile Ala Ala Arg Gly Arg Leu Pro Val Val Ala Gly Gly Ser Asn

165 170 175

Ser Leu Ile His Ala Leu Leu Ala Asp Arg Leu Asp Ala Gly Ala Ala

180 185 190

Asp Pro Phe Ser Ala Pro Pro Gln Pro Ala Pro Pro Arg Trp Gly Arg

195 200 205

Arg Pro Ala Leu Arg Ser Pro Cys Cys Leu Leu Trp Val His Val Asp

210 215 220

Ala Ala Leu Leu Ala Glu Tyr Leu Asp Arg Arg Val Asp Asp Met Val

225 230 235 240

Arg Gly Gly Met Val Glu Glu Leu Arg Glu Tyr Phe Ala Ala Thr Thr

245 250 255

Ala Ala Glu Arg Ala Ala His Ala Ala Gly Leu Gly Arg Ala Ile Gly

260 265 270

Val Pro Glu Leu Gly Ala Cys Phe Ala Gly Arg Ala Ser Phe Arg Ala

275 280 285

Ala Ile Asp Asp Ile Lys Ala Asn Thr Arg Asp Leu Ala Ala Ala Gln

290 295 300

Val Arg Lys Ile Arg Arg Met Ala Asp Ala Trp Gly Trp Pro Ile Gln

305 310 315 320

Arg Leu Asp Ala Ser Ala Thr Val Arg Ala Arg Leu Arg Gly Ala Gly

325 330 335

Pro Asp Ala Glu Ser Ala Cys Trp Glu Arg Asp Val Arg Ala Pro Gly

340 345 350

Leu Ala Ala Ile Arg Ser Phe Leu Leu Glu Leu Asp Gly Gly Ser Val

355 360 365

Val Asp Gly Ala Val Val Glu Glu Val Glu Pro Arg Val Arg Cys Cys

370 375 380

Asp Val Val Gly

385

<210>19

<211>1167

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT8 coding sequence

<400>19

atgaccaccc tcctcgccaa taggatcact acgctcgtgc gcgcccctcc tcctcccatg 60

gccgccgccg ccgtcgcggg agcgcggagg ccattgcacc ggaccttggc gcacccgcca 120

ccgcccgagg aggacgagca tcagcagcag cgcgcgtgcc gcagcagggg atcctcgtcc 180

tcctgctcgg cttcctcgtc atcgacgccc gcccggcccc ggggcacggg gatggtggtg 240

atcgtcggcg ccacgggcac cgggaagacc aagctgtcca tcgacgccgc ggaggcggtc 300

ggcggggagg tggtgaacgc ggataagatc cagctctacg ccgggctgga cgtgaccacg 360

aacaaggtgg cccccgcgga ccgccgcggc gtgccgcacc acctcctcgg cgccatccgc 420

cccgaggccg gcgagctccc gccctccacg ttccgctccc tcgccgccgc cacggccgcc 480

tcgatcgccg cgcgcggccg cctgccggtc gtcgcgggcg gctccaactc cctcatccac 540

gcgctcctcg ccgaccgcct cgacgccggc gccgccgacc ccttctccgc tccaccgcag 600

ccggcgccgc cgcggtgggg ccgccggccc gcgctccgat ccccgtgctg tctcctctgg 660

gtccacgtcg acgccgcgct cctcgcggag tacctggacc ggcgcgtgga cgacatggtg 720

cgcggcggca tggtggagga gctgcggggag tacttcgccg cgaccaccgc cgccgagcgc 780

gccgcgcacg ccgcggggct gggcagggcc atcggcgtgc ccgagctggg cgcctgcttc 840

gcggggcgcg ccagcttccg cgccgcgatc gacgacatca aggccaacac gcgggacctg 900

gcggccgcgc aggtgcgcaa gatccgacgc atggccgatg cctggggctg gcccatccag 960

cggctcgacg cgtcggccac agtccgcgcg cgcctccgcg gcgcggggcc cgacgcggag 1020

tcggcgtgct gggagcgcga cgtgcgcgcg cccgggctcg ccgccatccg gagcttcctt 1080

ctagagctgg acggcggcag cgtcgtcgac ggcgctgtgg tggaggaggt ggagccgcgg 1140

gtgcgatgct gcgacgtggt ggggtga 1167

<210>20

<211>1535

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT9 full-length sequence

<400>20

gcttgcttgt aaccgatggc actacttgca gagttcatac gtggctgcaa cccgatatca 60

agcagccatg aggcttccga aagtaagccg cctgtgtcaa ccagcatttc ttcacacctc 120

agatcaattg accggtacag ttcaatgcgt ggacttgcga ggaaaatgca gaagaagtca 180

tactccatct ccctggcctc gtagtttcca gcggatgagg gagcctcagt taggtcagtg 240

tcatgctgtt cacagaatgc atcgtacggt aaggcgaagg cagaaggagg ggagcctgat 300

gacctgataa tctcgaggct gcggctcaat ctgttccagt tattgacaga caagtcccgg 360

gctctggggt caccagcctg caccaccagc tctaccgctt cctcccactg gccgttctcc 420

cgaaaatcag cgagctcaga ccaacacggcc aaagtggact ccatggatga ctgtggaacg 480

ttcggcttgc catagatgta ccatctcagg tacagcccag tccctcctgc gataacgggc 540

acgcagcctc tgtcaagaac atcctgcgtc gccctccggg catcgcggaa aaaggctcca 600

gccgagtaat cgtcggacgt gtccagtatg tcgatcaagt gatgcggcac taggctcatc 660

tccgccgcgg acggtttggc agaaccaacg tcaaggccac ggtagacttg cacggagtcc 720

gcactgatga tctcccctcc gagcctcctg gccacctcca gcgccagcct gctctttcca 780

gcaccagtag gaccagagat gacgatgacc gtgtccttct tcttatgatg agttggtggt 840

ggcagagacg aggcggccat cgtggcggcc ttggttgagg cacagaaaga tggtagaagc 900

ctgtgctgtg actgcaagca caggataggc cagctcctcc agatggcacg ccgcatcgcc 960

cccagctgct gtggcctcat ctcgccggac atgataaagg aagagtatct gccaaagacg 1020

cagacagaaa ttcagggtag gctacgtgat ttgctgagat gaaaatgctg cttgacagcg 1080

tttgaagtcc tgcaggcatt tcatcataca cctagtagg actcagtcga gagccattgc 1140

tcctggcttg gctcggagtc atagagtggt agccttatt agccatcgtt agcaacaagg 1200

aagtggtgag tgtgaatgtg caggaagaag tacccatgga gaactgtatg tgaattcaaa 1260

cggaggatgc gtacttgtgg ctgtggctgc ggcggcgagc tccgacaagg ttgctctcgc 1320

aagctacaag gaggggagga gtgggggaac ggaagccatt ccgcttacct tgagccagca 1380

acttccggcc tctcctccgt cccaccgccg gcgtggcgtg gtgcgcctca gcgcctggcc 1440

gccgcgcttc cgaggacggc cagggagcag ccgtcggcgc ctcgggcgtt aggctatccg 1500

cactcatata actctaaatt tctactctaa aagaa 1535

<210>21

<211>3000

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmIPT1 genome sequence

<400>21

tttctgcaaa agtatctaag ttgattcttg ttaaagcctc tccttgattc ccaaatccag 60

catacccttg agagtctttt ctttagtcgg gtaagtcttg ctgagtaatt ccatactcag 120

ggttttattc cctgttgttt ttcaggtcat agttttgtgc tgttgatgat ggtgttaagt 180

gccggtgggc tcggccttct tataagtcta agtaaccctt ctaaacttct taatgaggat 240

ggtcccttga gctagcatat atttcaaact tatacttttg caatcactcc gataaaataa 300

tataaaattt ttgtaacttg taaaatttgg taacaaggtt ttcgctgcaa aaatattggt 360

gtgtgtgatt tgtgttactt aatcccgagg ttctggttgt aagtggttta tccggtgtcc 420

ttggggcaat cggacggatc ctgttaagtt atctggtgca catgcatagc agtctgaggt 480

ctttgagaca aggacaggtg catgtgggcc caataacttg ggaggttctg ccacaattat 540

tagcaagata tcggagatat ttatgtgcta tatttttact atagaggagt gagacgaaga 600

gtgttatgta agttacagag tagaaacaaa ttctactact gtataaaatc atttcacatc 660

ccccatccca tgaatttgag ataggcttat atctaaactt tggaaagtgg tggaatgtca 720

aattccaaac taaataagtt actttagtga gtgaattcca attcctttaa aatgaaggga 780

tccaaacgcc ccgtaaggaa aatagaaatc ccttaggctt tgtttgggta aagagagatt 840

gaagtggatt aaggtgtatt gaaggagatt aaaataaaaa ttagttcata ttacacttca 900

atacacctca taccacctca atccactcca atctgagatt acccaaacaa gtccttagta 960

aaattgtgtt cccaaactat gctctaattt tactagcatt ttttatccac taactattag 1020

ctccaaacac cccctaattt tagtagcaag agcaaggaaa accccccagc catcttcatc 1080

tgcctgctgg tgccggactg acggttagag atggcccacc cctcctccgc cgccgccgta 1140

tcctccacgg cgcccgctgc aaaccctagt tatggcgccc gcgaggaagg aggcgcccgc 1200

tctccgccgt ctccgtctcc gtctccgtct cagagggggc gggccaaggt ggtgatcgtt 1260

atgggcgcca cgggcgccgg caagtcgcgg ctggccgtcg acctcgcggc ccacttcgcc 1320

ggcgtcgagg tggtcagcgc cgactccatg cagctctacc gcggcctcga cgtcctcacc 1380

aacaaggctc ccctccacga gcagaacggt ctgtttctga ctcctcaccg cctcccccct 1440

aatttcagtt tcctgtcaga ttaaatgctc gagcctgttc catgcgtgtt tgcaggtgtt 1500

cctcatcatc tacttagcgt gattgatccc tctgtcgagt tcacttgccg tgatttccgc 1560

gaccgtgccc tgccggtaag ccagtgctgc tgccagccac tgcctctaca agttccagca 1620

cttgctttag ttggtcacat gatagctaag gccttcccct ctgctcacag attatacagg 1680

aaatagtgga ccgcggtggc ctccctgtgg ttgtcggcgg cacaaacttc tacatccagg 1740

ttcaaatttg aagtgtccta atttctgtat ggttttctgg gtgaccgcag tgtctgaacc 1800

accgtcgtgt ttttgcccac aaaatactcc aggctctcgt tagcccattc ctcttggatg 1860

atatggcaga agaaatgcag ggctgtactc tgagagatca catagatgat ggtataggcg 1920

agtaatgagt catgctaaat gttccttgtt cttagtatga acaatcagcg gttgacatgt 1980

atgccaatcg tcaggcctta ccgatgaaga tgaaggcaat gggtttgaac gcttgaagga 2040

gatcgatcct gtggctgcgc agaggatcca tccaaacgac catagaaaag taagggtgtt 2100

gcccaagttt ttcctgactt tatgcagctc ttgagctcta ccatgttaga attacktctt 2160

gtacaggtgc tcattttcct actgccgttg attgcttgtg tatttttcgt tctgcatggg 2220

ttggtgtatc acaaagatca aacgctacct cgagttgtat gcaaccacgg gtgccctacc 2280

cagcgatctg ttccaaggag aggccgctaa ggtgaggaat ttttcgctgc acttgtctgc 2340

tctgcttgta taatatcctc cctttgtcat gaacatgagc cgacaattgc ctcacaaggc 2400

tcttcttttt ttggttttgt ttccctaatt gagtataaac ttgctgggaa ttcctgctca 2460

tcatgtcaga acagaaatgg ggtcggccta gtaactccag actcgactgc tgtttcctgt 2520

gggtagatgc tgatcttcaa gtcctggaca gttatgtcaa caaaagggtc gattgcatga 2580

tggatggtgg cctgctggac gaagtatgca gcatatatga tgcggatgct gtctataccc 2640

aggggctgcg gcaggctatt ggggttcgtg agtttgacga gtttttcaga gcatatttac 2700

ccagaaaaga atctggtgag ggttcctgtg caagcctgtt aggtatgcat gacgatcagc 2760

ttaagagctt gttggacgaa gctgtttccc agctgaaggc aaacactcgt agactagttc 2820

gacgtcaagt aagcacttag tatcctgtgt atttttttat gttgttgtgt tgttttagaa 2880

tactgtgcga ctgaacacac ggatacttgg ctcaatgtag acttcttagc gtttcttttt 2940

ttttctccat taaataaaag agacggagat tgcatcggct gagtaaagat tttgggtgga 3000

<210>22

<211>1209

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT1 full length (from ZmlPT1 genomic clone)

<221> CDS

<222>(1)...(1062)

<400>22

atg gcc cac ccc tcc tcc gcc gcc gcc gta tcc tcc acg gcg ccc gct 48

Met Ala His Pro Ser Ser Ala Ala Ala Val Ser Ser Thr Ala Pro Ala

1 5 10 15

gca aac cct agt tat ggc gcc cgc gag gaa gga ggc gcc cgc tct ccg 96

Ala Asn Pro Ser Tyr Gly Ala Arg Glu Glu Gly Gly Ala Arg Ser Pro

20 25 30

ccg tct ccg tct ccg tct ccg tct cag agg ggg cgg gcc aag gtg gtg 144

Pro Ser Pro Ser Pro Ser Pro Ser Gln Arg Gly Arg Ala Lys Val Val

35 40 45

atc gtt atg ggc gcc acg ggc gcc ggc aag tcg cgg ctg gcc gtc gac 192

Ile Val Met Gly Ala Thr Gly Ala Gly Lys Ser Arg Leu Ala Val Asp

50 55 60

ctc gcg gcc cac ttc gcc ggc gtc gag gtg gtc agc gcc gac tcc atg 240

Leu Ala Ala His Phe Ala Gly Val Glu Val Val Ser Ala Asp Ser Met

65 70 75 80

cag ctc tac cgc ggc ctc gac gtc ctc acc aac aag gct ccc ctc cac 288

Gln Leu Tyr Arg Gly Leu Asp Val Leu Thr Asn Lys Ala Pro Leu His

85 90 95

gag cag aac ggt gtt cct cat cat cta ctt agc gtg att gat ccc tct 336

Glu Gln Asn Gly Val Pro His His Leu Leu Ser Val Ile Asp Pro Ser

100 105 110

gtc gag ttc act tgc cgt gat ttc cgc gac cgt gcc ctg ccg att ata 384

Val Glu Phe Thr Cys Arg Asp Phe Arg Asp Arg Ala Leu Pro Ile Ile

115 120 125

cag gaa ata gtg gac cgc ggt ggc ctc cct gtg gtt gtc ggc ggc acs 432

Gln Glu Ile Val Asp Arg Gly Gly Leu Pro Val Val Val Gly Gly Thr

130 135 140

aac ttc tac atc cag gct ctc gtt agc cca ttc ctc ttg gat gat atg 480

Asn Phe Tyr Ile Gln Ala Leu Val Ser Pro Phe Leu Leu Asp Asp Met

145 150 155 160

gca gaa gaa atg cag ggc tgt act ctg aga gat cac ata gat gat ggc 528

Ala Glu Glu Met Gln Gly Cys Thr Leu Arg Asp His Ile Asp Asp Gly

165 170 175

ctt acc gat gaa gat gaa ggc aat ggg ttt gaa cgc ttg aag gag atc 576

Leu Thr Asp Glu Asp Glu Gly Asn Gly Phe Glu Arg Leu Lys Glu Ile

180 185 190

gat cct gtg gct gcg cag agg atc cat cca aac gac cat aga aaa atc 624

Asp Pro Val Ala Ala Gln Arg Ile His Pro Asn Asp His Arg Lys Ile

195 200 205

aaa cgc tac ctc gag ttg tat gca acc acg ggt gcc cta ccc agc gat 672

Lys Arg Tyr Leu Glu Leu Tyr Ala Thr Thr Gly Ala Leu Pro Ser Asp

210 215 220

ctg ttc caa gga gag gcc gct aag aaa tgg ggt cgg cct agt aac tcc 720

Leu Phe Gln Gly Glu Ala Ala Lys Lys Trp Gly Arg Pro Ser Asn Ser

225 230 235 240

aga ctc gac tgc tgt ttc ctg tgg gta gat gct gat ctt caa gtc ctg 768

Arg Leu Asp Cys Cys Phe Leu Trp Val Asp Ala Asp Leu Gln Val Leu

245 250 255

gac agt tat gtc aac aaa agg gtc gat tgc atg atg gat ggt ggc ctg 816

Asp Ser Tyr Val Asn Lys Arg Val Asp Cys Met Met Asp Gly Gly Leu

260 265 270

ctg gac gaa gta tgc agc ata tat gat gcg gat gct gtc tat acc cag 864

Leu Asp Glu Val Cys Ser Ile Tyr Asp Ala Asp Ala Val Tyr Thr Gln

275 280 285

ggg ctg cgg cag gct att ggg gtt cgt gag ttt gac gag ttt ttc aga 912

Gly Leu Arg Gln Ala Ile Gly Val Arg Glu Phe Asp Glu Phe Phe Arg

290 295 300

gca tat tta ccc aga aaa gaa tct ggt gag ggt tcc tgt gca agc ctg 960

Ala Tyr Leu Pro Arg Lys Glu Ser Gly Glu Gly Ser Cys Ala Ser Leu

305 310 315 320

tta ggt atg cat gac gat cag ctt aag agc ttg ttg gac gaa gct gtt 1008

Leu Gly Met His Asp Asp Gln Leu Lys Ser Leu Leu Asp Glu Ala Val

325 330 335

tcc cag ctg aag gca aac act cgt aga cta gtt cga cgt caa gta agc 1056

Ser Gln Leu Lys Ala Asn Thr Arg Arg Leu Val Arg Arg Gln Val Ser

340 345 350

act tag tatcctgtgt atttttttat gttgttgtgt tgttttagaa tactgtgcga 1112

Thr *

ctgaacacac ggatacttgg ctcaatgtag acttcttagc gtttcttttt ttttctccat 1172

taaataaaag agacggagat tgcatcggct gagtaaa 1209

<210>23

<211>353

<212>PRT

<213> corn

<400>23

Met Ala His Pro Ser Ser Ala Ala Ala Val Ser Ser Thr Ala Pro Ala

1 5 10 15

Ala Asn Pro Ser Tyr Gly Ala Arg Glu Glu Gly Gly Ala Arg Ser Pro

20 25 30

Pro Ser Pro Ser Pro Ser Pro Ser Gln Arg Gly Arg Ala Lys Val Val

35 40 45

Ile Val Met Gly Ala Thr Gly Ala Gly Lys Ser Arg Leu Ala Val Asp

50 55 60

Leu Ala Ala His Phe Ala Gly Val Glu Val Val Ser Ala Asp Ser Met

65 70 75 80

Gln Leu Tyr Arg Gly Leu Asp Val Leu Thr Asn Lys Ala Pro Leu His

85 90 95

Glu Gln Asn Gly Val Pro His His Leu Leu Ser Val Ile Asp Pro Ser

100 105 110

Val Glu Phe Thr Cys Arg Asp Phe Arg Asp Arg Ala Leu Pro Ile Ile

115 120 125

Gln Glu Ile Val Asp Arg Gly Gly Leu Pro Val Val Val Gly Gly Thr

130 135 140

Asn Phe Tyr Ile Gln Ala Leu Val Ser Pro Phe Leu Leu Asp Asp Met

145 150 155 160

Ala Glu Glu Met Gln Gly Cys Thr Leu Arg Asp His Ile Asp Asp Gly

165 170 175

Leu Thr Asp Glu Asp Glu Gly Asn Gly Phe Glu Arg Leu Lys Glu Ile

180 185 190

Asp Pro Val Ala Ala Gln Arg Ile His Pro Asn Asp His Arg Lys Ile

195 200 205

Lys Arg Tyr Leu Glu Leu Tyr Ala Thr Thr Gly Ala Leu Pro Ser Asp

210 215 220

Leu Phe Gln Gly Glu Ala Ala Lys Lys Trp Gly Arg Pro Ser Asn Ser

225 230 235 240

Arg Leu Asp Cys Cys Phe Leu Trp Val Asp Ala Asp Leu Gln Val Leu

245 250 255

Asp Ser Tyr Val Asn Lys Arg Val Asp Cys Met Met Asp Gly Gly Leu

260 265 270

Leu Asp Glu Val Cys Ser Ile Tyr Asp Ala Asp Ala Val Tyr Thr Gln

275 280 285

Gly Leu Arg Gln Ala Ile Gly Val Arg Glu Phe Asp Glu Phe Phe Arg

290 295 300

Ala Tyr Leu Pro Arg Lys Glu Ser Gly Glu Gly Ser Cys Ala Ser Leu

305 310 315 320

Leu Gly Met His Asp Asp Gln Leu Lys Ser Leu Leu Asp Glu Ala Val

325 330 335

Ser Gln Leu Lys Ala Asn Thr Arg Arg Leu Val Arg Arg Gln Val Ser

340 345 350

Thr

<210>24

<211>1062

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT1 coding sequence

<400>24

atggccccacc cctcctccgc cgccgccgta tcctccacgg cgcccgctgc aaaccctagt 60

tatggcgccc gcgaggaagg aggcgcccgc tctccgccgt ctccgtctcc gtctccgtct 120

cagaggggggc gggccaaggt ggtgatcgtt atgggcgcca cgggcgccgg caagtcgcgg 180

ctggccgtcg acctcgcggc ccacttcgcc ggcgtcgagg tggtcagcgc cgactccatg 240

cagctctacc gcggcctcga cgtcctcacc aacaaggctc ccctccacga gcagaacggt 300

gttcctcatc atctacttag cgtgattgat ccctctgtcg agttcacttg ccgtgatttc 360

cgcgaccgtg ccctgccgat tatacaggaa atagtggacc gcggtggcct ccctgtggtt 420

gtcggcggca caaacttcta catccaggct ctcgttagcc cattcctctt ggatgatatg 480

gcagaagaaa tgcagggctg tactctgaga gatcacatag atgatggcct taccgatgaa 540

gatgaaggca atgggtttga acgcttgaag gagatcgatc ctgtggctgc gcagaggatc 600

catccaaacg accatagaaa aatcaaacgc tacctcgagt tgtatgcaac cacgggtgcc 660

ctacccagcg atctgttcca aggagaggcc gctaagaaat gggtcggcc tagtaactcc 720

agactcgact gctgtttcct gtgggtagat gctgatcttc aagtcctgga cagttatgtc 780

aacaaaaggg tcgattgcat gatggatggt ggcctgctgg acgaagtatg cagcatatat 840

gatgcggatg ctgtctatac ccaggggctg cggcaggcta ttggggttcg tgagtttgac 900

gagtttttca gagcatattt accccagaaaa gaatctggtg agggttcctg tgcaagcctg 960

ttaggtatgc atgacgatca gcttaagagc ttgttggacg aagctgtttc ccagctgaag 1020

gcaaacactc gtagactagt tcgacgtcaa gtaagcactt ag 1062

<210>25

<211>1082

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> ZmlPT1 promoter

<400>25

tttctgcaaa agtatctaag ttgattcttg ttaaagcctc tccttgattc ccaaatccag 60

catacccttg agagtctttt ctttagtcgg gtaagtcttg ctgagtaatt ccatactcag 120

ggttttattc cctgttgttt ttcaggtcat agttttgtgc tgttgatgat ggtgttaagt 180

gccggtgggc tcggccttct tataagtcta agtaaccctt ctaaacttct taatgaggat 240

ggtcccttga gctagcatat atttcaaact tatacttttg caatcactcc gataaaataa 300

tataaaattt ttgtaacttg taaaatttgg taacaaggtt ttcgctgcaa aaatattggt 360

gtgtgtgatt tgtgttactt aatcccgagg ttctggttgt aagtggttta tccggtgtcc 420

ttggggcaat cggacggatc ctgttaagtt atctggtgca catgcatagc agtctgaggt 480

ctttgagaca aggacaggtg catgtgggcc caataacttg ggaggttctg ccacaattat 540

tagcaagata tcggagatat ttatgtgcta tatttttact atagaggagt gagacgaaga 6O0

gtgttatgta agttacagag tagaaacaaa ttctactact gtataaaatc atttcacatc 660

ccccatccca tgaatttgag ataggcttat atctaaactt tggaaagtgg tggaatgtca 720

aattccaaac taaataagtt actttagtga gtgaattcca attcctttaa aatgaaggga 780

tccaaacgcc ccgtaaggaa aatagaaatc ccttaggctt tgtttgggta aagagagatt 840

gaagtggatt aaggtgtatt gaaggagatt aaaataaaaa ttagttcata ttacacttca 900

atacacctca taccacctca atccactcca atctgagatt acccaaacaa gtccttagta 960

aaattgtgtt cccaaactat gctctaattt tactagcatt ttttatccac taactattag 1020

ctccaaacac cccctaattt tagtagcaag agcaaggaaa accccccagc catcttcatc 1080

tg 1082

<210>26

<211>1299

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> Variant of ZmlPT1, full length, from B73

EST

<221> CDS

<222>(31)...(1086)

<400>26

ccacgcgtcc ggccggactg acggttagag atg gcc cac ccc tcc gcc gcc gcc 54

                                                                       , 

1 5

gcc gcc gta tcc tcc acg gcg ccc gct gca aac cct agt tct ggc gcc 102

Ala Ala Val Ser Ser Thr Ala Pro Ala Ala Asn Pro Ser Ser Gly Ala

10 15 20

cgc gag gaa gga ggc gcc cgc tct ccg ccg tcg ccg tct ccg tct cag 150

Arg Glu Glu Gly Gly Ala Arg Ser Pro Pro Ser Pro Ser Pro Ser Gln

25 30 35 40

agg ggg cgg gcc aag gtg gtg atc gtt atg ggc gcc acg ggc gcc ggc 198

Arg Gly Arg Ala Lys Val Val Ile Val Met Gly Ala Thr Gly Ala Gly

45 50 55

aag tcg cgg ctg gcc gtc gac ctc gcg gcc cac ttc gcc ggc gtc gaa 246

Lys Ser Arg Leu Ala Val Asp Leu Ala Ala His Phe Ala Gly Val Glu

60 65 70

gtg gtc agc gcc gac tcc atg cag ctc tac cgc ggc ctc gac gtc ctc 294

Val Val Ser Ala Asp Ser Met Gln Leu Tyr Arg Gly Leu Asp Val Leu

75 80 85

acc aac aag gct ccc ctc cac gag cag aac ggt gtt cct cat cat cta 342

Thr Asn Lys Ala Pro Leu His Glu Gln Asn Gly Val Pro His His Leu

90 95 100

ctt agc gtg att gat ccc tct gtc gag ttc act tgc cgt gat ttc cgc 390

Leu Ser Val Ile Asp Pro Ser Val Glu Phe Thr Cys Arg Asp Phe Arg

105 110 115 120

gac cgt gcc gtg ccg att ata cag gaa ata gtg gac cgc ggt ggc ctc 438

Asp Arg Ala Val Pro Ile Ile Gln Glu Ile Val Asp Arg Gly Gly Leu

125 130 135

cct gtg gtt gtc ggc ggc aca aac ttc tac atc cag gct ctc gtt agc 486

Pro Val Val Val Gly Gly Thr Asn Phe Tyr Ile Gln Ala Leu Val Ser

140 145 150

cca ttc ctc ttg gat gat atg gca gaa gaa atg cag ggc tgt act ctg 534

Pro Phe Leu Leu Asp Asp Met Ala Glu Glu Met Gln Gly Cys Thr Leu

155 160 165

aga gat cac ata gat gat ggt ctt act gat gaa gat gaa ggc aat ggg 582

Arg Asp His Ile Asp Asp Gly Leu Thr Asp Glu Asp Glu Gly Asn Gly

170 175 180

ttt gaa cgc ttg aag gag atc gat cct gtg gct gcg cag agg atc cat 630

Phe Glu Arg Leu Lys Glu Ile Asp Pro Val Ala Ala Gln Arg Ile His

185 190 195 200

cca aac gac cat aga aaa atc aaa cgc tac ctc gag ttg tat gca acc 678

Pro Asn Asp His Arg Lys Ile Lys Arg Tyr Leu Glu Leu Tyr Ala Thr

205 210 215

acg ggt gcc cta ccc agc gat ctg ttc cas gga gag gcc gct aag aaa 726

Thr Gly Ala Leu Pro Ser Asp Leu Phe Gln Gly Glu Ala Ala Lys Lys

220 225 230

tgg ggt cgg cct agt aac tcc aga ctc gac tgc tgt ttc ctg tgg gta 774

Trp Gly Arg Pro Ser Asn Ser Arg Leu Asp Cys Cys Phe Leu Trp Val

235 240 245

gat gct gat ctt caa gtc ctg gac agt tat gtc aac aaa agg gtc gat 822

Asp Ala Asp Leu Gln Val Leu Asp Ser Tyr Val Asn Lys Arg Val Asp

250 255 260

tgc atg atg gat ggt ggc ctg ctg gac gaa gta tgc agc ata tat gat 870

Cys Met Met Asp Gly Gly Leu Leu Asp Glu Val Cys Ser Ile Tyr Asp

265 270 275 280

gcg gat gct gtc tat acc cag ggg ctg cgg cag gct att ggg gtt cgt 918

Ala Asp Ala Val Tyr Thr Gln Gly Leu Arg Gln Ala Ile Gly Val Arg

285 290 295

gag ttt gac gag ttt ttc aga gca tat tta ccc aga aaa gaa tct ggt 966

Glu Phe Asp Glu Phe Phe Arg Ala Tyr Leu Pro Arg Lys Glu Ser Gly

300 305 310

gag ggt tcc tgt gca agc ctg tta ggt atg cat gac gat cag ctt aag 1014

Glu Gly Ser Cys Ala Ser Leu Leu Gly Met His Asp Asp Gln Leu Lys

315 320 325

agc ttg ttg gac gaa gct gtt tcc cag ctg aag gca aac act cgt aga 1062

Ser Leu Leu Asp Glu Ala Val Ser Gln Leu Lys Ala Asn Thr Arg Arg

330 335 340

cta gtt cga cgt caa gta agc act tagtatcctg tgtatttttt tatgttgttg 1116

Leu Val Arg Arg Gln Val Ser Thr

345 350

tgttgtttta gaatactgtg cgactgaaca cacggatact tggctcaatg tagacttctt 1176

agcgtttctt tttttttctc cattaaataa aagagacgga gattgcatcg gctgagtaaa 1236

aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1296

aaa 1299

<210>27

<211>352

<212>PRT

<213> corn

<400>27

Met Ala His Pro Ser Ala Ala Ala Ala Ala Val Ser Ser Thr Ala Pro

1 5 10 15

Ala Ala Asn Pro Ser Ser Gly Ala Arg Glu Glu Gly Gly Ala Arg Ser

20 25 30

Pro Pro Ser Pro Ser Pro Ser Gln Arg Gly Arg Ala Lys Val Val Ile

35 40 45

Val Met Gly Ala Thr Gly Ala Gly Lys Ser Arg Leu Ala Val Asp Leu

50 55 60

Ala Ala His Phe Ala Gly Val Glu Val Val Ser Ala Asp Ser Met Gln

65 70 75 80

Leu Tyr Arg Gly Leu Asp Val Leu Thr Asn Lys Ala Pro Leu His Glu

85 90 95

Gln Asn Gly Val Pro His His Leu Leu Ser Val Ile Asp Pro Ser Val

100 105 110

Glu Phe Thr Cys Arg Asp Phe Arg Asp Arg Ala Val Pro Ile Ile Gln

115 120 125

Glu Ile Val Asp Arg Gly Gly Leu Pro Val Val Val Gly Gly Thr Asn

130 135 140

Phe Tyr Ile Gln Ala Leu Val Ser Pro Phe Leu Leu Asp Asp Met Ala

145 150 155 160

Glu Glu Met Gln Gly Cys Thr Leu Arg Asp His Ile Asp Asp Gly Leu

165 170 175

Thr Asp Glu Asp Glu Gly Asn Gly Phe Glu Arg Leu Lys Glu Ile Asp

180 185 190

Pro Val Ala Ala Gln Arg Ile His Pro Asn Asp His Arg Lys Ile Lys

195 200 205

Arg Tyr Leu Glu Leu Tyr Ala Thr Thr Gly Ala Leu Pro Ser Asp Leu

210 215 220

Phe Gln Gly Glu Ala Ala Lys Lys Trp Gly Arg Pro Ser Asn Ser Arg

225 230 235 240

Leu Asp Cys Cys Phe Leu Trp Val Asp Ala Asp Leu Gln Val Leu Asp

245 250 255

Ser Tyr Val Asn Lys Arg Val Asp Cys Met Met Asp Gly Gly Leu Leu

260 265 270

Asp Glu Val Cys Ser Ile Tyr Asp Ala Asp Ala Val Tyr Thr Gln Gly

275 280 285

Leu Arg Gln Ala Ile Gly Val Arg Glu Phe Asp Glu Phe Phe Arg Ala

290 295 300

Tyr Leu Pro Arg Lys Glu Ser Gly Glu Gly Ser Cys Ala Ser Leu Leu

305 310 315 320

Gly Met His Asp Asp Gln Leu Lys Ser Leu Leu Asp Glu Ala Val Ser

325 330 335

Gln Leu Lys Ala Asn Thr Arg Arg Leu Val Arg Arg Gln Val Ser Thr

340 345 350

<210>28

<211>1056

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

<223> variant ZmlPT1, cds, from B73 EST

<400>28

atggcccacc cctccgccgc cgccgccgcc gtatcctcca cggcgcccgc tgcaaaccct 60

agttctggcg cccgcgagga aggaggcgcc cgctctccgc cgtcgccgtc tccgtctcag 120

aggggggcggg ccaaggtggt gatcgttatg ggcgccacgg gcgccggcaa gtcgcggctg 180

gccgtcgacc tcgcggccca cttcgccggc gtcgaagtgg tcagcgccga ctccatgcag 240

ctctaccgcg gcctcgacgt cctcaccaac aaggctcccc tccacgagca gaacggtgtt 300

cctcatcatc tacttagcgt gattgatccc tctgtcgagt tcacttgccg tgatttccgc 360

gaccgtgccg tgccgattat acaggaaata gtggaccgcg gtggcctccc tgtggttgtc 420

ggcggcacaa acttctacat ccaggctctc gttagcccat tcctcttgga tgatatggca 480

gaagaaatgc agggctgtac tctgagagat cacatagatg atggtcttac tgatgaagat 540

gaaggcaatg ggtttgaacg cttgaaggag atcgatcctg tggctgcgca gaggatccat 600

ccaaacgacc atagaaaaat caaacgctac ctcgagttgt atgcaaccac gggtgcccta 660

cccagcgatc tgttccaagg agaggccgct aagaaatggg gtcggcctag taactccaga 720

ctcgactgct gtttcctgtg ggtagatgct gatcttcaag tcctggacag ttatgtcaac 780

aaaagggtcg attgcatgat ggatggtggc ctgctggacg aagtatgcag catatatgat 840

gcggatgctg tctataccca ggggctgcgg caggctattg gggttcgtga gtttgacgag 900

tttttcagag catatttacc cagaaaagaa tctggtgagg gttcctgtgc aagcctgtta 960

ggtatgcatg acgatcagct taagagcttg ttggscgaag ctgtttccca gctgaaggca 1020

aacactcgta gactagttcg acgtcasgta agcact 1056

<210>29

<211>357

<212>PRT

<213> Arabidopsis thaliana

<400>29

Met Thr Glu Leu Asn Phe His Leu Leu Pro Ile Ile Ser Asp Arg Phe

1 5 10 15

Thr Thr Thr Thr Thr Thr Thr Ser Pro Ser Phe Ser Ser His Ser Ser Ser Ser

20 25 30

Ser Ser Ser Leu Leu Ser Phe Thr Lys Arg Arg Arg Lys His Gln Pro

35 40 45

Leu Val Ser Ser Ile Arg Met Glu Gln Ser Arg Ser Arg Asn Arg Lys

50 55 60

Asp Lys Val Val Val Ile Leu Gly Ala Thr Gly Ala Gly Lys Ser Arg

65 70 75 80

Leu Ser Val Asp Leu Ala Thr Arg Phe Pro Ser Glu Ile Ile Asn Ser

85 90 95

Asp Lys Ile Gln Val Tyr Glu Gly Leu Glu Ile Thr Thr Asn Gln Ile

100 105 110

Thr Leu Gln Asp Arg Arg Gly Val Pro His His Leu Leu Gly Val Ile

115 120 125

Asn Pro Glu His Gly Glu Leu Thr Ala Gly Glu Phe Arg Ser Ala Ala

130 135 140

Ser Asn Val Val Lys Glu Ile Thr Ser Arg Gln Lys Val Pro Ile Ile

145 150 155 160

Ala Gly Gly Ser Asn Ser Phe Val His Ala Leu Leu Ala Gln Arg Phe

165 170 175

Asp Pro Lys Phe Asp Pro Phe Ser Ser Gly Ser Cys Leu Ile Ser Ser

180 185 190

Asp Leu Arg Tyr Glu Cys Cys Phe Ile Trp Val Asp Val Ser Glu Thr

195 200 205

Val Leu Tyr Glu Tyr Leu Leu Arg Arg Val Asp Glu Met Met Asp Ser

210 215 220

Gly Met Phe Glu Glu Leu Ser Arg Phe Tyr Asp Pro Val Lys Ser Gly

225 230 235 240

Leu Glu Thr Arg Phe Gly Ile Arg Lys Ala Ile Gly Val Pro Glu Phe

245 250 255

Asp Gly Tyr Phe Lys Glu Tyr Pro Pro Glu Lys Lys Met Ile Lys Trp

260 265 270

Asp Ala Leu Arg Lys Ala Ala Tyr Asp Lys Ala Val Asp Asp Ile Lys

275 280 285

Arg Asn Thr Trp Thr Leu Ala Lys Arg Gln Val Lys Lys Ile Glu Met

290 295 300

Leu Lys Asp Ala Gly Trp Glu Ile Glu Arg Val Asp Ala Thr Ala Ser

305 310 315 320

Phe Lys Ala Val Met Met Lys Ser Ser Ser Glu Lys Lys Trp Arg Glu

325 330 335

Asn Trp Glu Glu Gln Val Leu Glu Pro Ser Val Lys Ile Val Lys Arg

340 345 350

His Leu Val Gln Asn

355

<210>30

<211>318

<212>PRT

<213> Arabidopsis

<400>30

Met Lys Cys Asn Asp Lys Met Val Val Ile Met Gly Ala Thr Gly Ser

1 5 10 15

Gly Lys Ser Ser Leu Ser Val Asp Leu Ala Leu His Phe Lys Ala Glu

20 25 30

Ile Ile Asn Ser Asp Lys Met Gln Phe Tyr Asp Gly Leu Lys Ile Thr

35 40 45

Thr Asn Gln Ser Thr Ile Glu Asp Arg Arg Gly Val Pro His His Leu

50 55 60

Leu Gly Glu Leu Asn Pro Glu Ala Gly Glu Val Thr Ala Ala Glu Phe

65 70 75 80

Arg Val Met Ala Ala Glu Ala Ile Ser Glu Ile Thr Gln Arg Lys Lys

85 90 95

Leu Pro Ile Leu Ala Gly Gly Ser Asn Ser Tyr Ile His Ala Leu Leu

100 105 110

Ala Lys Ser Tyr Asp Pro Glu Asn Tyr Pro Phe Ser Asp His Lys Gly

115 120 125

Ser Ile Cys Ser Glu Leu Lys Tyr Asp Cys Cys Phe Ile Trp Ile Asp

130 135 140

Val Asp Gln Ser Val Leu Phe Glu Tyr Leu Ser Leu Arg Leu Asp Leu

145 150 155 160

Met Met Lys Ser Gly Met Phe Glu Glu Ile Ala Glu Phe His Arg Ser

165 170 175

Lys Lys Ala Pro Lys Glu Pro Leu Gly Ile Trp Lys Ala Ile Gly Val

180 185 190

Gln Glu Phe Asp Asp Tyr Leu Lys Met Tyr Lys Trp Asp Asn Asp Met

195 200 205

Asp Lys Trp Asp Pro Met Arg Lys Glu Ala Tyr Glu Lys Ala Val Arg

210 215 220

Ala Ile Lys Glu Asn Thr Phe Gln Leu Thr Lys Asp Gln Ile Thr Lys

225 230 235 240

Ile Asn Lys Leu Arg Asn Ala Gly Trp Asp Ile Lys Lys Val Asp Ala

245 250 255

Thr Ala Ser Phe Arg Glu Ala Ile Arg Ala Ala Lys Glu Gly Glu Gly

260 265 270

Val Ala Glu Met Gln Arg Lys Ile Trp Asn Lys Glu Val Leu Glu Pro

275 280 285

Cys Val Lys Ile Val Arg Ser His Leu Asp Gln Pro Ile Asn Tyr Tyr

290 295 300

Tyr Tyr Tyr Phe Tyr Leu Leu Lys Arg Phe Leu Ser Leu Asn

305 310 315

<210>31

<211>350

<212>PRT

<213>Petunia x hybrida

<400>31

Met Leu Ile Val Val His Ile Ile Ser Ile Thr Arg Ile Ile Phe Ile

1 5 10 15

Thr Leu Thr His Asn His Leu His Phe Leu Met Phe Arg Ser Leu Ser

20 25 30

Tyr Asn His Lys His Leu Lys Phe Leu Thr Asn Pro Thr Thr Arg Val

35 40 45

Leu Arg Arg Asn Met Ser Ser Ser Ser Thr Val Val Thr Ile Pro Gly Pro

50 55 60

Thr Gln Lys Asn Lys Asn Lys Ile Ile Val Ile Met Gly Ala Thr Gly

65 70 75 80

Ser Gly Lys Ser Lys Leu Ser Ile Asp Leu Val Thr Arg His Tyr Pro

85 90 95

Phe Ser Glu Ile Ile Asn Ser Asp Lys Ile Gln Ile Thr Lys Gly Leu

100 105 110

Asn Ile Thr Thr Asn Lys Ile Thr Val Pro Asp Arg Arg Gly Val Val

115 120 125

His His Leu Leu Gly Glu Ile Asp Pro Asp Phe Asn Phe Ser Pro Ser

130 135 140

His Phe Arg Ser Ile Ala Gly Gln Arg Ile Asn Ser Ile Ile Asn Arg

145 150 155 160

His Lys Leu Pro Phe Leu Val Gly Gly Ser Asn Ser Tyr Ile Tyr Ala

165 170 175

Leu Leu Thr Asn Arg Phe Asp Pro Asp Phe Asn Pro Asp Ser Asn Pro

180 185 190

Val His Phe Ile Ser Asn Glu Leu Arg Tyr Asn Cys Cys Phe Ile Trp

195 200 205

Val Asp Val Leu Asn Pro Val Leu Asn Glu Tyr Leu Asp Lys Arg Val

210 215 220

Asp Glu Met Met Asn Ser Gly Met Tyr Glu Glu Leu Glu Gln Phe Phe

225 230 235 240

Lys Glu Asn Arg Phe Ser Asp Pro Gly Leu Glu Pro Gly Arg Ala Thr

245 250 255

Gly Leu Arg Lys Ala Ile Gly Val Pro Glu Met Glu Arg Tyr Phe Lys

260 265 270

Lys Ser Cys Thr Tyr Glu Glu Ala Val Arg Glu Ile Lys Glu Asn Thr

275 280 285

Trp Arg Leu Ala Lys Lys Gln Met Trp Lys Ile Gln Arg Leu Arg Glu

290 295 300

Ala Gly Trp Asp Leu Gln Arg Val Asp Ala Thr Glu Ala Phe Val Glu

305 310 315 320

Ala Met Ser Asn Lys Lys Glu Lys Gly Ile Ile Trp Glu Lys Gln Val

325 330 335

Val Glu Pro Ser Val Lys Ile Val Asn Arg Phe Leu Leu Asp

340 345 350

<210>32

<211>92

<212>PRT

<213> Artificial sequence

<220>

<223> Cytokinin synthase consensus sequence

<221> variant

<222>(8)...(8)

<223> The amino acid at position 8 can be T.

<221> variant

<222>(14)...(14)

<223> The amino acid at position 14 can be L or I.

<221> variant

<222>(22)...(23)

<223> Amino acids at positions 22 and 23 can be L or I.

<221> variant

<222>(87)...(88)

<223> Amino acids at positions 87 and 88 can be L or I.

<221> variant

<222>(92)...(92)

<223> The amino acid at position 92 can be T.

<221> variant

<222>(2)...(89)

<223> Xaa = any amino acid

<400>32

Gly Xaa Thr Xaa Xaa Gly Lys Ser Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Val Val Xaa Xaa Asp Xaa Xaa Gln Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

65 70 75 80

Xaa Xaa Xaa Xaa Xaa Xaa Val Val Xaa Gly Gly Ser

85 90

<210>33

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> Consensus zinc finger motifs found in tRNA IPTs

<221> variant

<222>2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19,

20, 21, 22

<223> Xaa = any amino acid

<400>33

Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

His Xaa Xaa Xaa Xaa Xaa His

20

<210>34

<211>336

<212>PRT

<213> Arabidopsis

<400>34

Met Ile Met Lys Ile Ser Met Ala Met Cys Lys Gln Pro Leu Pro Pro

1 5 10 15

Ser Pro Thr Leu Asp Phe Pro Pro Ala Arg Phe Gly Pro Asn Met Leu

20 25 30

Thr Leu Asn Pro Tyr Gly Pro Lys Asp Lys Val Val Val Ile Met Gly

35 40 45

Ala Thr Gly Thr Gly Lys Ser Arg Leu Ser Val Asp Ile Ala Thr Arg

50 55 60

Phe Arg Ala Glu Ile Ile Asn Ser Asp Lys Ile Gln Val His Gln Gly

65 70 75 80

Leu Asp Ile Val Thr Asn Lys Ile Thr Ser Glu Glu Ser Cys Gly Val

85 90 95

Pro His His Leu Leu Gly Val Leu Pro Pro Glu Ala Asp Leu Thr Ala

100 105 110

Ala Asn Tyr Cys His Met Ala Asn Leu Ser Ile Glu Ser Val Leu Asn

115 120 125

Arg Gly Lys Leu Pro Ile Ile Val Gly Gly Ser Asn Ser Tyr Val Glu

130 135 140

Ala Leu Val Asp Asp Lys Glu Asn Lys Phe Arg Ser Arg Tyr Asp Cys

145 150 155 160

Cys Phe Leu Trp Val Asp Val Ala Leu Pro Val Leu His Gly Phe Val

165 170 175

Ser Glu Arg Val Asp Lys Met Val Glu Ser Gly Met Val Glu Glu Val

180 185 190

Arg Glu Phe Phe Asp Phe Ser Asn Ser Asp Tyr Ser Arg Gly Ile Lys

195 200 205

Lys Ala Ile Gly Phe Pro Glu Phe Asp Arg Phe Phe Arg Asn Glu Gln

210 215 220

Phe Leu Asn Val Glu Asp Arg Glu Glu Leu Leu Ser Lys Val Leu Glu

225 230 235 240

Glu Ile Lys Arg Asn Thr Phe Glu Leu Ala Cys Arg Gln Arg Glu Lys

245 250 255

Ile Glu Arg Leu Arg Lys Val Lys Lys Trp Ser Ile Gln Arg Val Asp

260 265 270

Ala Thr Pro Val Phe Thr Lys Arg Arg Ser Lys Met Asp Ala Asn Val

275 280 285

Ala Trp Glu Arg Leu Val Ala Gly Pro Ser Thr Asp Thr Val Ser Arg

290 295 300

Phe Leu Leu Asp Ile Ala Ser Arg Arg Pro Leu Val Glu Ala Ser Thr

305 310 315 320

Ala Val Ala Ala Ala Met Glu Arg Glu Leu Ser Arg Cys Leu Val Ala

325 330 335

<210>35

<211>330

<212>PRT

<213> Arabidopsis

<400>35

Met Lys Pro Cys Met Thr Ala Leu Arg Gln Val Ile Gln Pro Leu Ser

1 5 10 15

Leu Asn Phe Gln Gly Asn Met Val Asp Val Pro Phe Phe Arg Arg Lys

20 25 30

Asp Lys Val Val Phe Val Met Gly Ala Thr Gly Thr Gly Lys Ser Arg

35 40 45

Leu Ala Ile Asp Leu Ala Thr Arg Phe Pro Ala Glu Ile Val Asn Ser

50 55 60

Asp Lys Ile Gln Val Tyr Lys Gly Leu Asp Ile Val Thr Asn Lys Val

65 70 75 80

Thr Pro Glu Glu Ser Leu Gly Val Pro His His Leu Leu Gly Thr Val

85 90 95

His Asp Thr Tyr Glu Asp Phe Thr Ala Glu Asp Phe Gln Arg Glu Ala

100 105 110

Ile Arg Ala Val Glu Ser Ile Val Gln Arg Asp Arg Val Pro Ile Ile

115 120 125

Ala Gly Gly Ser Asn Ser Tyr Ile Glu Ala Leu Val Asn Asp Cys Val

130 135 140

Asp Phe Arg Leu Arg Tyr Asn Cys Cys Phe Leu Trp Val Asp Val Ser

145 150 155 160

Arg Pro Val Leu His Ser Phe Val Ser Glu Arg Val Asp Lys Met Val

165 170 175

Asp Met Gly Leu Val Asp Glu Val Arg Arg Ile Phe Asp Pro Ser Ser

180 185 190

Ser Asp Tyr Ser Ala Gly Ile Arg Arg Ala Ile Gly Val Pro Glu Leu

195 200 205

Asp Glu Phe Leu Arg Ser Glu Met Arg Asn Tyr Pro Ala Glu Thr Thr

210 215 220

Glu Arg Leu Leu Glu Thr Ala Ile Glu Lys Ile Lys Glu Asn Thr Cys

225 230 235 240

Leu Leu Ala Cys Arg Gln Leu Gln Lys Ile Gln Arg Leu Tyr Lys Gln

245 250 255

Trp Lys Trp Asn Met His Arg Val Asp Ala Thr Glu Val Phe Leu Arg

260 265 270

Arg Gly Glu Glu Ala Asp Glu Ala Trp Asp Asn Ser Val Ala His Pro

275 280 285

Ser Ala Leu Ala Val Glu Lys Phe Leu Ser Tyr Ser Asp Asp His His

290 295 300

Leu Glu Gly Ala Asn Ile Leu Leu Pro Glu Ile Ser Ala Val Pro Pro

305 310 315 320

Leu Pro Ala Ala Val Ala Ala Ile Ser Arg

325 330

<210>36

<211>342

<212>PRT

<213> Arabidopsis

<400>36

Met Gln Gln Leu Met Thr Leu Leu Ser Pro Pro Leu Ser His Ser Ser

1 5 10 15

Leu Leu Pro Thr Val Thr Thr Lys Phe Gly Ser Pro Arg Leu Val Thr

20 25 30

Thr Cys Met Gly His Ala Gly Arg Lys Asn Ile Lys Asp Lys Val Val

35 40 45

Leu Ile Thr Gly Thr Thr Gly Thr Gly Lys Ser Arg Leu Ser Val Asp

50 55 60

Leu Ala Thr Arg Phe Phe Pro Ala Glu Ile Ile Asn Ser Asp Lys Met

65 70 75 80

Gln Ile Tyr Lys Gly Phe Glu Ile Val Thr Asn Leu Ile Pro Leu His

85 90 95

Glu Gln Gly Gly Val Pro His His Leu Leu Gly Gln Phe His Pro Gln

100 105 110

Asp Gly Glu Leu Thr Pro Ala Glu Phe Arg Ser Leu Ala Thr Leu Ser

115 120 125

Ile Ser Lys Leu Ile Ser Ser Lys Lys Leu Pro Ile Val Val Gly Gly

130 135 140

Ser Asn Ser Phe Asn His Ala Leu Leu Ala Glu Arg Phe Asp Pro Asp

145 150 155 160

Ile Asp Pro Phe Ser Pro Gly Ser Ser Leu Ser Thr Ile Cys Ser Asp

165 170 175

Leu Arg Tyr Lys Cys Cys Ile Leu Trp Val Asp Val Leu Glu Pro Val

180 185 190

Leu Phe Gln His Leu Cys Asn Arg Val Asp Gln Met Ile Glu Ser Gly

195 200 205

Leu Val Glu Gln Leu Ala Glu Leu Tyr Asp Pro Val Val Asp Ser Gly

210 215 220

Arg Arg Leu Gly Val Arg Lys Thr Ile Gly Val Glu Glu Phe Asp Arg

225 230 235 240

Tyr Phe Arg Val Tyr Pro Lys Glu Met Asp Lys Gly Ile Trp Asp Leu

245 250 255

Ala Arg Lys Ala Ala Tyr Glu Glu Thr Val Lys Gly Met Lys Glu Arg

260 265 270

Thr Cys Arg Leu Val Lys Lys Gln Lys Glu Lys Ile Met Lys Leu Ile

275 280 285

Arg Gly Gly Trp Glu Ile Lys Arg Leu Asp Ala Thr Ala Ala Ile Met

290 295 300

Ala Glu Leu Asn Gln Ser Thr Ala Lys Gly Glu Gly Lys Asn Gly Arg

305 310 315 320

Glu Ile Trp Glu Lys His Ile Val Asp Glu Ser Val Glu Ile Val Lys

325 330 335

Lys Phe Leu Leu Glu Val

340

<210>37

<211>329

<212>PRT

<213> Arabidopsis

<400>37

Met Lys Phe Ser Ile Ser Ser Ser Leu Lys Gln Val Gln Pro Ile Leu Cys

1 5 10 15

Phe Lys Asn Lys Leu Ser Lys Val Asn Val Asn Ser Phe Leu His Pro

20 25 30

Lys Glu Lys Val Ile Phe Val Met Gly Ala Thr Gly Ser Gly Lys Ser

35 40 45

Arg Leu Ala Ile Asp Leu Ala Thr Arg Phe Gln Gly Glu Ile Ile Asn

50 55 60

Ser Asp Lys Ile Gln Leu Tyr Lys Gly Leu Asp Val Leu Thr Asn Lys

65 70 75 80

Val Thr Pro Lys Glu Cys Arg Gly Val Pro His His Leu Leu Gly Val

85 90 95

Phe Asp Ser Glu Ala Gly Asn Leu Thr Ala Thr Gln Tyr Ser Arg Leu

100 105 110

Ala Ser Gln Ala Ile Ser Lys Leu Ser Ala Asn Asn Lys Leu Pro Ile

115 120 125

Val Ala Gly Gly Ser Asn Ser Tyr Ile Glu Ala Leu Val Asn His Ser

130 135 140

Ser Gly Phe Leu Leu Asn Asn Tyr Asp Cys Cys Phe Ile Trp Val Asp

145 150 155 160

Val Ser Leu Pro Val Leu Asn Ser Phe Val Ser Lys Arg Val Asp Arg

165 170 175

Met Met Glu Ala Gly Leu Leu Glu Glu Val Arg Glu Val Phe Asn Pro

180 185 190

Lys Ala Asn Tyr Ser Val Gly Ile Arg Arg Ala Ile Gly Val Pro Glu

195 200 205

Leu His Glu Tyr Leu Arg Asn Glu Ser Leu Val Asp Arg Ala Thr Lys

210 215 220

Ser Lys Met Leu Asp Val Ala Val Lys Asn Ile Lys Lys Asn Thr Glu

225 230 235 240

Ile Leu Ala Cys Arg Gln Leu Lys Lys Ile Gln Arg Leu His Lys Lys

245 250 255

Trp Lys Met Ser Met His Arg Val Asp Ala Thr Glu Val Phe Leu Lys

260 265 270

Arg Asn Val Glu Glu Gln Asp Glu Ala Trp Glu Asn Leu Val Ala Arg

275 280 285

Pro Ser Glu Arg Ile Val Asp Lys Phe Tyr Asn Asn Asn Asn Gln Leu

290 295 300

Lys Asn Asp Asp Val Glu His Cys Leu Ala Ala Ser Tyr Gly Gly Gly

305 310 315 320

Ser Gly Ser Arg Ala His Asn Met Ile

325

<210>38

<211>330

<212>PRT

<213> Arabidopsis

<400>38

Met Gln Asn Leu Thr Ser Thr Phe Val Ser Pro Ser Met Ile Pro Ile

1 5 10 15

Thr Ser Pro Arg Leu Arg Leu Pro Pro Pro Arg Ser Val Val Pro Met

20 25 30

Thr Thr Val Cys Met Glu Gln Ser Tyr Lys Gln Lys Val Val Val Ile

35 40 45

Met Gly Ala Thr Gly Ser Gly Lys Ser Cys Leu Ser Ile Asp Leu Ala

50 55 60

Thr Arg Phe Ser Gly Glu Ile Val Asn Ser Asp Lys Ile Gln Phe Tyr

65 70 75 80

Asp Gly Leu Lys Val Thr Thr Asn Gln Met Ser Ile Leu Glu Arg Cys

85 90 95

Gly Val Pro His His Leu Leu Gly Glu Leu Pro Pro Asp Asp Ser Glu

100 105 110

Leu Thr Thr Ser Glu Phe Arg Ser Leu Ala Ser Arg Ser Ile Ser Glu

115 120 125

Ile Thr Ala Arg Gly Asn Leu Pro Ile Ile Ala Gly Gly Ser Asn Ser

130 135 140

Phe Ile His Ala Leu Leu Val Asp Arg Phe Asp Pro Lys Thr Tyr Pro

145 150 155 160

Phe Ser Ser Glu Thr Ser Ile Ser Ser Gly Leu Arg Tyr Glu Cys Cys

165 170 175

Phe Leu Trp Val Asp Val Ser Val Ser Val Leu Phe Glu Tyr Leu Ser

180 185 190

Lys Arg Val Asp Gln Met Met Glu Ser Gly Met Phe Glu Glu Leu Ala

195 200 205

Gly Phe Tyr Asp Pro Arg Tyr Ser Gly Ser Ala Ile Arg Ala His Gly

210 215 220

Ile His Lys Thr Ile Gly Ile Pro Glu Phe Asp Arg Tyr Phe Ser Leu

225 230 235 240

Tyr Pro Pro Glu Arg Lys Gln Lys Met Ser Glu Trp Asp Gln Ala Arg

245 250 255

Lys Gly Ala Tyr Asp Glu Ala Val Gln Glu Ile Lys Glu Asn Thr Trp

260 265 270

Arg Leu Ala Lys Lys Gln Ile Glu Arg Ile Met Lys Leu Lys Ser Ser

275 280 285

Gly Trp Asp Ile Gln Arg Leu Asp Ala Thr Pro Ser Phe Gly Arg Ser

290 295 300

Ser Arg Glu Ile Trp Asp Asn Thr Val Leu Asp Glu Ser Ile Lys Val

305 310 315 320

Val Lys Arg Phe Leu Val Lys Asp Lys Val

325 330

<210>39

<211>350

<212>PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of ZmlPT1

<400>39

Met Ala His Pro Ala Ala Ala Ala Ala Val Ser Ser Thr Ala Pro Ala

1 5 10 15

Ala Asn Pro Ser Gly Ala Arg Glu Glu Glu Gly Gly Ala Arg Ser Pro Pro

20 25 30

Ser Pro Ser Pro Ser Gln Arg Gly Arg Ala Lys Val Val Ile Val Met

35 40 45

Gly Ala Thr Gly Ala Gly Lys Ser Arg Leu Ala Val Asp Leu Ala Ala

50 55 60

His Phe Ala Gly Val Glu Val Val Ser Ala Asp Ser Met Gln Leu Tyr

65 70 75 80

Arg Gly Leu Asp Val Leu Thr Asn Lys Ala Pro Leu His Glu Gln Asn

85 90 95

Gly Val Pro His His Leu Leu Ser Val Ile Asp Pro Ser Val Glu Phe

100 105 110

Thr Cys Arg Asp Phe Arg Asp Arg Ala Leu Pro Ile Ile Gln Glu Ile

115 120 125

Val Asp Arg Gly Gly Leu Pro Val Val Val Gly Gly Thr Asn Phe Tyr

130 135 140

Ile Gln Ala Leu Val Ser Pro Phe Leu Leu Asp Asp Met Ala Glu Glu

145 150 155 160

Met Gln Gly Cys Thr Leu Arg Asp His Ile Asp Asp Gly Leu Thr Asp

165 170 175

Glu Asp Glu Gly Asn Gly Phe Glu Arg Leu Lys Glu Ile Asp Pro Val

180 185 190

Ala Ala Gln Arg Ile His Pro Asn Asp His Arg Lys Ile Lys Arg Tyr

195 200 205

Leu Glu Leu Tyr Ala Thr Thr Gly Ala Leu Pro Ser Asp Leu Phe Gln

210 215 220

Gly Glu Ala Ala Lys Lys Trp Gly Arg Pro Ser Asn Ser Arg Leu Asp

225 230 235 240

Cys Cys Phe Leu Trp Val Asp Ala Asp Leu Gln Val Leu Asp Ser Tyr

245 250 255

Val Asn Lys Arg Val Asp Cys Met Met Asp Gly Gly Leu Leu Asp Glu

260 265 270

Val Cys Ser Ile Tyr Asp Ala Asp Ala Val Tyr Thr Gln Gly Leu Arg

275 280 285

Gln Ala Ile Gly Val Arg Glu Phe Asp Glu Phe Phe Arg Ala Tyr Leu

290 295 300

Pro Arg Lys Glu Ser Gly Glu Gly Ser Cys Ala Ser Leu Leu Gly Met

305 310 315 320

His Asp Asp Gln Leu Lys Ser Leu Leu Asp Glu Ala Val Ser Gln Leu

325 330 335

Lys Ala Asn Thr Arg Arg Leu Val Arg Arg Gln Val Ser Thr

340 345 350

<210>40

<211>4680

<212>DNA

<213> Rice (Oryza sativa)

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT8 genome sequence (017718_1)

<221> exon

<222>(225)...(489)

<221> exon

<222>(576)...(655)

<221> exon

<222>(801)...(869)

<221> exon

<222>(966)...(1047)

<221> exon

<222>(1158)...(1252)

<221> exon

<222>(1385)...(1459)

<221> exon

<222>(1641)...(1692)

<221> exon

<222>(1698)...(1886)

<221> exon

<222>(1923)...(2008)

<221> exon

<222>(2083)...(2158)

<221> exon

<222>(2257)...(2420)

<221> exon

<222>(2563)...(2682)

<400>40

accatagtga aagaaaaaat acgttggagt acgccgattt tcttgtaaat ttgatatttc 60

actcccctag tccccttatta ccaatttttt tttttaaaaa aagaaaaaac gcaaccttac 120

cagcccaaag gcccaaagcc cacaacagag aggagagtgg gcgacgggggg gctagggcgg 180

cggcggcgcg gcgaccagcg acggcggcg cgcacctgac cggaatggcc cacctcgcgg 240

cctctgccgc cccgcttcca agcgctgacc ccgacgccgg cgaggagtcc tcccactctc 300

cgccgccgcc ggagaagggg ctgaggaagg tggtggtggt gatgggcgcg actggcgccg 360

gcaagtcgcg gctggccgtc gacctcgcga gccacttcgc cggcgtcgag gtggtcagcg 420

ccgactccat gcaagtctac ggtgggctcg atgtcctcac caacaaggtc cccctccacg 480

agcagaaagg tctcctcccg ggattcccca gttcttcttt tgaccaaacc tgcttcagat 540

cgagcttaac agcgctatct ttgccgtgtt accaggcgtt cctcaccatc tcctgagcgt 600

gattgatccc tctgtggagt tcacttgccg cgatttccgc gaccatgctg tgccggtgag 660

cctatgatgt tgctgctacg acttttagtg ctcctagtgt gccatgttta ctgattagtt 720

gatgtttctt agtgctctgc tcaccaatta tataaggtat attggtttac tgattaattg 780

ctgtttctga gtggtcacag atttagaag gtatattgga tcgtggcggc ctccctgtta 840

ttgttggtgg tacaaacttc tacatccagg ttgatactta agcgcatgag gattcctgta 900

tattaggcta tttttcttct gaattagact atatctgatt tttgtccttt taacacttat 960

tgtaggctct tgttagccca ttcctctttg atgatatggc acaggatatt gagggtctta 1020

ctttaaatga ccacctagat gagataggtg aatgatgaaa gcttagcaca tgtttcttgt 1080

tgttagcatg ttttgatcaa tggttgtgtc caattagtgt ttgacttgtt aaacactgct 1140

taacacatgc caagcagggc ttgataatga tgatgaagcc ggtctgtatg aacatttgaa 1200

gaagattgat cctgttgctg cacaaaggat acacccgaac aaccatcgaa aagtaagggt 1260

gttgcacagt tgtgccctta acctgttagg tttctttggt agcaattgga ttttccttgt 1320

ggtgttgccc catttgcctt atccggttat cctgttctgc atgctttttt gttgtgttga 1380

ccagataaaa cgctaccttg agttgtatga atccacaggt gccctacta gtgatctttt 1440

ccaagggcaa gccacagagg tgagaaaaaa atgatttccc ttttaattaa tttctttatt 1500

ctgacttgtt gctgactcta tagtccatgt gaaatgtgca aggactttat gcatattatc 1560

atgcgcacaa cacatttttt gccgtacgag ttggacctca tgcgaactct aaatgtccta 1620

atgaggtcat ttgttgtcag gacagaagtg gggtcgacct agtaactcca gatttgactg 1680

ttgtttcttg tggttagatg ctgatcttca tgttctggat cgttatgtca atgaaagggt 1740

cgactgcatg attgatgatg gcctgctaga tgaagtgtgt aacatatg atcgagaggc 1800

cacttatacc caagggctgc ggcaggccat tggtgttcgt gaatttgatg agtttttcag 1860

attttatttt gcaaggaagg aaaccggtga gataaagatg gattcctgta caactatggc 1920

aggtctccat gatgataacc tgaagggctt attggatgaa gcagtctcac aactaaaagc 1980

aaacactcgc agacttgttc gacgtcaagt aatctcgaca cttttttaag taaataattg 2040

aaaattgcat tttgtgtgtt ttatattctt gcctttcttc agagacgaag gctgcatcgg 2100

ttgaataaat attttgagtg gaacttgcgt catattgatg caacagaagc tttctatggt 2160

aatgatatgt gcatttcatg ttttagttca aagccaaaag atttcatgtc ttacgaaatc 2220

taatgtgttt gcttaacatg tcatgcatat ttctaggtgc cactgctgac tcatggaaca 2280

tgaaagttgt gaaaccttgc gtggatattg ttagagattt cttgtctgat gatacaattt 2340

tggcaagcag agatggttct agtgtaactg gaagccctag gatgtcttca agagagttgt 2400

ggactcaata tgtttgtgag gtaattggga ggcttttctt attcttacca aaaagaatgt 2460

tgataactgt atcgtcattt gtgcgttttg ccacattttt tgttagtggg acagcaatca 2520

atctgatgaa actttcttgc ctttcctgct cctattttac aggcctgtga taaccgggta 2580

cttcggggaa cgcatgagtg ggagcaacac aagcaaggcc gatgccaccg taaaagagta 2640

caacgtttga aacagaaggc tagtacagtg atatcattta aggcaattag cactgtttgc 2700

actctcggtg ttcatgaacc tttcttcatt ctctgcaact gtccccatgc atcctgtttg 2760

tcaaattggc tgaagactac accattcaga aggtagcaag cagcagatat atttgttaat 2820

agtaccttgc tagattcttg tgccagttcc aaacatccaa tgcagagaat acaaactcta 2880

cagattggtc agcacaagca cgtccgattg agcagcatct acactgatga ccagttggag 2940

tttctccaat ctgctgatca tttctagact agttttccca ttaaggacac cataaattgg 3000

gtaggcggtc cagcttgtta gcaaagtggt gatagtgatt agcaattaag catgacattg 3060

acccatcgaa tatttgcata tcttggtctt ccagattgca tgatttttcc ttcatatgtg 3120

actggaaaca gtggggccat gctaggttac ataaattcct gggcgtgata cactgcgaat 3180

agtagctatc atgtttacta ctgtcgtgtt gagactactg tacagtagct cgtatgtatt 3240

tctcgtatgt ttgtgcataa gtgaggggtc gatgagagtg acttactaga cttttctcat 3300

cctaaattcc taataactag aaaagatgac cgaaattggg aaggcgactt gtgcctcttt 3360

tggaatgatc gaaatataga ggaactttca tgttgacctg attcttacga aaatcatgta 3420

aaactcgtgt tcgttgtcaa aaggcccaac ttcatctcag atgagcataa gtataccata 3480

ttaatgcttc aaaatggtta atgctagctc gtttttactg cacaactaat gctcgatgtc 3540

caaatatact tgggtttatta ttattttttt gaaggatttt tcatgtgagt ctcgccgagg 3600

tccactaacc ggtacacagg cgccgacctc tggcacatta ttttacacga gaaatttaag 3660

gtaggcatga aatcatcagt cgcacggatg caaacgtgac gacatcatca gaaacaatat 3720

actgctgcgc cgatttaaac tacacttaaa ttaaataatt ctattagtgg tacgagagta 3780

gtactactcc tgtatgtaga atagatgtgc acgggcgcac gtgtttcatc cctctaattc 3840

tgaatcccca cgtgacgatc gagcttaaag ccgaacgggc ggggcggggg gataaagcgg 3900

gtcccccagc cgctgtctcc agttcacacc cacaacccga agtcgatcgc tcgtgttcgt 3960

gtccgcctcg acggcgaact cgacgggtcc cgacccgcas acccaacacc caaccctsct 4020

tatacccacc tccactaatc cctcctctca tcgcaccacc acgccactga gctcaagcta 4080

agctaagtgc taacctaggt gttcgaccat ggacaccgag gacacgtcgt cggcttcgtc 4140

ctcgtcggtg tcgccgccgt cgtcgccggg cggcgggcac caccaccggc tgccgccgaa 4200

gcggcgggcg gggcggaaga aattccggga gacgcggcac ccggtgtacc gcggcgtgcg 4260

cgcgcgggcg ggggggagca ggtgggtgtg cgaggtgcgc gagccgcagg cgcaggcgcg 4320

catctggctc ggcacctacc cgacgccgga gatggcggcg cgcgcgcacg acgtcgcggc 4380

catcgccctc cgcggcgagc gcggcgccga gctcaacttc ccggactccc cctccacgct 4440

cccgcgcgcg cgcacggcgt cgcccgagga catccgcctc gccgccgcgc aggccgccga 4500

gctgtaccgc cgcccgccgc cgccgctggc attgccggag gatccgcagg aaggcacgag 4560

cggcggcggc gccaccgcca cctcggggcg tccggctgcc gtgttcgtgg acgaggacgc 4620

catcttcgac atgccggggc tgatcgacga catggcgagg gggatgatgc tgacgccgcc 4680

<210>41

<211>450

<212>PRT

<213> Rice

<220>

<221>CHAIN

<222>(0)...(0)

<223> OsIPT8 amino acid sequence (017718_1)

<400>41

Met Ala His Leu Ala Ala Ser Ala Ala Pro Leu Pro Ser Ala Asp Pro

1 5 10 15

Asp Ala Gly Glu Glu Ser Ser Ser His Ser Pro Pro Pro Pro Glu Lys Gly

20 25 30

Leu Arg Lys Val Val Val Val Met Gly Ala Thr Gly Ala Gly Lys Ser

35 40 45

Arg Leu Ala Val Asp Leu Ala Ser His Phe Ala Gly Val Glu Val Val

50 55 60

Ser Ala Asp Ser Met Gln Val Tyr Gly Gly Leu Asp Val Leu Thr Asn

65 70 75 80

Lys Val Pro Leu His Glu Gln Lys Gly Val Pro His His Leu Leu Ser

85 90 95

Val Ile Asp Pro Ser Val Glu Phe Thr Cys Arg Asp Phe Arg Asp His

100 105 110

Ala Val Pro Ile Ile Glu Gly Ile Leu Asp Arg Gly Gly Leu Pro Val

115 120 125

Ile Val Gly Gly Thr Asn Phe Tyr Ile Gln Ala Leu Val Ser Pro Phe

130 135 140

Leu Phe Asp Asp Met Ala Gln Asp Ile Glu Gly Leu Thr Leu Asn Asp

145 150 155 160

His Leu Asp Glu Ile Gly Leu Asp Asn Asp Asp Glu Ala Gly Leu Tyr

165 170 175

Glu His Leu Lys Lys Ile Asp Pro Val Ala Ala Gln Arg Ile His Pro

180 185 190

Asn Asn His Arg Lys Ile Lys Arg Tyr Leu Glu Leu Tyr Glu Ser Thr

195 200 205

Gly Ala Leu Pro Ser Asp Leu Phe Gln Gly Gln Ala Thr Glu Asp Arg

210 215 220

Ser Gly Val Asp Leu Val Thr Pro Asp Leu Thr Val Val Ser Cys Asp

225 230 235 240

Ala Asp Leu His Val Leu Asp Arg Tyr Val Asn Glu Arg Val Asp Cys

245 250 255

Met Ile Asp Asp Gly Leu Leu Asp Glu Val Cys Asn Ile Tyr Asp Arg

260 265 270

Glu Ala Thr Tyr Thr Gln Gly Leu Arg Gln Ala Ile Gly Val Arg Glu

275 280 285

Phe Asp Glu Phe Phe Arg Phe Tyr Phe Ala Arg Lys Glu Thr Gly Leu

290 295 300

His Asp Asp Asn Leu Lys Gly Leu Leu Asp Glu Ala Val Ser Gln Leu

305 310 315 320

Lys Ala Asn Thr Arg Arg Leu Val Arg Arg Gln Arg Arg Arg Leu His

325 330 335

Arg Leu Asn Lys Tyr Phe Glu Trp Asn Leu Arg His Ile Asp Ala Thr

340 345 350

Glu Ala Phe Tyr Gly Ala Thr Ala Asp Ser Trp Asn Met Lys Val Val

355 360 365

Lys Pro Cys Val Asp Ile Val Arg Asp Phe Leu Ser Asp Asp Thr Ile

370 375 380

Leu Ala Ser Arg Asp Gly Ser Ser Val Thr Gly Ser Pro Arg Met Ser

385 390 395 400

Ser Arg Glu Leu Trp Thr Gln Tyr Val Cys Glu Ala Cys Asp Asn Arg

405 410 415

Val Leu Arg Gly Thr His Glu Trp Glu Gln His Lys Gln Gly Arg Cys

420 425 430

His Arg Lys Arg Val Gln Arg Leu Lys Gln Lys Ala Ser Thr Val Ile

435 440 445

Ser Leu

450

<210>42

<211>8463

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT11 genome sequence (006475_2)

<221> exon

<222>(2001)...(2834)

<221> exon

<222>(3574)...(3597)

<221> exon

<222>(5549)...(6463)

<400>42

tttgttgtga acttgagagg aataagttca tttgtgttcc ctcaacttga cgtcgggttc 60

gaattacgtc cccaaaccac aatacaatat aacacatcct caacttgcaa tacaggctca 120

tattaggtcc caaaacagta ctataccctag ttttggctga tgtagcgcac acgtaactca 180

tttgactagg tcctcatctc acgtggcatt gacatggtgc ttacgtagca attcgacaga 240

taaaataatt aaaactatgg ggctcgcata tcaatagtgg ggccatgcgg ggtccacatg 300

tcaataatag aaatggaaaa acaaaatggg cccacttgtc atccccgtcc atttctcgcc 360

atccccttcc ctatcttcgg caacaatgag ggaggcggct ggaggaggag ctcaagcggc 420

cgctagcaag tgcggcagct gcggcgggcg cgacaacagt tgatgacgat agggtcatcc 480

gctctcctct cctcccctcc catccctggc caccgccgtc ctcccttacc cccggcgctg 540

actaggtcta ctctgcggcc atggccttct ccccgaccat ccacctccac cacccacatt 600

gcctcctcct ctctccaacc ccttgtctat ctccccctac cacccatggc tctccgcctg 660

atcgagcccc cgccgccatg acccccttcg tcgtccctcc tctactctcg acatcctcct 720

gagcccttcc cctcctttga cttcggatga ggatgcacgg gcttggagcg ggaggtggag 780

agggcaagga gctcgcttgc tccactctgg ctgcttcacc caccgctcgc tccactctgc 840

ccttctcctc gcaggggaga gaaaggggga acatagcgat gctcacaaaa atgcgtttaa 900

acgcatttaa ttctggttta ggtacttgat gagtgaggat gacatgtggg acccacatgg 960

tcccaccgct ttttaattat tttgtggtgt aactgacaag tgatcccacg gttttattat 1020

ttttctcgga tcaaattgcc acgtaagcac cagagacaat attaccacgg acatcttttg 1080

tattggtttt gtaagctaag ggatgtgttg tatctggttt tgcggttaaa gacgaaattc 1140

aaaatgagcg cgactaaata agggacctaa agtgaactat tccaacttgt gatcctgacc 1200

aggaatcacc tggtccatat tgggccgacc cgaaccagat taaagcggat aacttctatg 1260

cttcatcatg tatactagcc acatgtgccc gcgcttcgct gcggatcatg aatttgtaaa 1320

ttaccatagt aaagaaagtt ttttctaata tgtatactga tccattgttt aaatggttgc 1380

catatatttt attacktaat gataccccac gcgttgttgc agaaaattct caaattttag 1440

ttatgggta gaatgtacaa cctaatgcta aaaagttaga gataaacaaa atgatttttt 1500

gaccaataat tgtgtgaata gcgtaagtca aattctaaaa ataatttagg taacacattt 1560

tagcagaaac ttcaaaagat gtacatactt tgaggtcatg agttaacata caatgttgta 1620

ctgtattatt catcaaatat tgtcacaatg tagcttactc gtagatataa gattgtatgt 1680

atctagtgag ggacaattta tattgttcac taacatctaa tattgttctc ggaatggtat 1740

ccagagaaat ttttatgatt tagaataaaa tggaacaatg ttgtataaat ctataaaaat 1800

ataattgcat aattatttat atgcttagat ttggcacctc taaatgtggc ctaactacca 1860

ttggcaccaca gttagtaggg gctcataaag atgcatcccc tataaaagcc aagggacacc 1920

gagagtcctc tacggaagaa ttccaccctt cccattagga cagtcaaaca ccttattgct 1980

accccaatct tcctttcagt atggagaact cctcaaagaa aacccaagag ttcttcccta 2040

aaggtgggaa tggaggttat gctgagcagc tggagctctt gctgaagcag cttcgttttc 2100

ctaacaagcc gatccaccat gcggagcaag tgatcaaagg attccggaag gattggacga 2160

tgaagatcta cattcaagcc agggaagaga agtgtcaagg acatgtgttc aagtcccgcc 2220

accttcgagc caacaaagag gcagcactcc aggatgcgtc gcgtgaggca ttcatgcgtc 2280

tatgtaagat ctacagcatc gaggttgcaa gtactccgtt ctttctacat ccattccgtg 2340

aatgcggtga ccgccgctgc catattcgga aatttagggg ctttgaggag cagtcgccca 2400

tccacttctc catgtggatg tgggctgcag acgaggccta tgaggaggcc ttagaggaat 2460

tagatatgct tcggtcaaag atcgccggct gggaggagcg gtacaaccac cttgctaaag 2520

aacacaccac tcgtggacaa ctattggaag caatcaagct tcgcctccag tggtattttc 2580

gaaccccatc tcaagctcat atccaacgga ctttgccacc accacccacaa agagtgacaa 2640

gaagtgatgg tgaggactat agtcaaatca atgcacatca ggcatgtctg gaaaggtccg 2700

aagttaaact tgatagggca acttcacaag actatctgca aggatacaag cccccatcag 2760

aatccctcga cgctattgtt tggcctcttg ttgaagggaa gcatgacaat acaagcagtg 2820

gtagggaggaa tgaggtaaag gaaactgctc acaataacca agggaccttg ttgggctagt 2880

cctcggaaag agagttggac cagctacata tctagaagac tgctatgtaa gtgataggtg 2940

gctatatctt gtcacgtagg agtagcatgt gggtgggagt tggaccaatt tcacatatag 3000

gagaccgcta tgtaagtgac aggttatggc ctgtcaccta gcagtactat gtgggtggtc 3060

aagatcacct atcaagtgtg cttgtctatg cctagttgtg gcctaccagt tagagtagta 3120

tgtgagggtg gtagtaagat tgcattccct ttgtccagtt gtgggtggac aagctaggcg 3180

gtagtctag tgtgtttatg tatgcgtggt tgtgatgctt ttgtgcttgg cccgaggaca 3240

ttgagcaata tttgcttaaa aatgcttgtt ttcttctgca atgctacttt gttttcatga 3300

tcatgcaagt tacctaaata catgtgaatt gttctagttg atgggatcta ttgcgataga 3360

atcacatgat ttccaattgt atagtaacgg agctagcaac agtaatataa ccattttgac 3420

caggatggtt caaaagtaaa ccatatagaa aaggagttgt ttattaaata tatgtattgt 3480

atcaactaaa atagtacaca atggccaata attttgcaat gaatttagtt tataattggc 3540

atggtatggt tatttttttt ttgcattttg cagaaggcat gggaaatggc aaaacaagta 3600

aatatataac aaagtaattt ctaacgattg ttagtaaccg gaagatggtt ggtattagat 3660

taccaagttt ggaagtatta ttttaccaga gaacgtataa gtaacatgta tattgttcga 3720

agtgcccaca tttgaattta cgttcgatga agaattgtta tgtaattttt ccttgaaaaa 3780

tgtgcaaaag cacatgttta caaatcatca ccatatctta agatgaaagt aggcataagg 3840

tttaaaaagt caaaggtaat tattaggttt atttttttgt tatgcttaca cacgtattga 3900

cgatacaaag gttcgagcca ttaactctga tgcctaaaaa tatctacaaa gaaaaaaaaa 3960

tcgatcacaa ttgcttgaat aatagtaaga gtatacctaa ctgaatttag ggcccaccaa 4020

tataattact gtacacatcc aactgcacgt ggattgatat gccaaattac tataatcgga 4080

ggtgcctaga ggaaggattt atcctttggt ctataaacat caagacaata ttggaccggt 4140

cctcgaaaag agagttggac cagctgcaca tctaggagac cgctatgtaa gtgacagggt 4200

catatcacat ctaggagacc tctaagtaag tgacagggtc atatcttgtc acctaggagt 4260

agtatgtggg tgagagttgg accaacttca catataggta accgctatgt aagtgatagg 4320

gtcatagcct gtcacctagt agcactatgt gggtgatcaa aatggcctct ctggtgtgct 4380

tgtctatgcc taattgtaat gcttttgtgc ttggctcgag gccaatgagt aatgtatgct 4440

tgaactgcta tgtaagcgac agggtcatag cctgttagtt agagtagtag gtgagggtgg 4500

taataaaatt gtattccctt tgtctagtta tgggtggaca aggtgggtaa tctagtgtgt 4560

ttgtgtatgc gtggttgtga tgcttttgtg cttggcccga ggataatgag caatatttgc 4620

ttaaagttgc atgttttctt ctccaatgca ggtttgtttt catgagcatg caaattatct 4680

aaatacatgc gaattgttct agttgatggg atctattgcg atagaatcaa atggcctcta 4740

atcgtatagt aacagagcta gcagtaatat aaccatctta accaggatgg ttcaaaagca 4800

agccatat aaaaggagtt gtttattaaa tatatgtatt gtacaaactg gaatattaca 4860

caagggctaa taattttgca atgaatttat ttgataattg gcattgtatg gctattttgt 4920

tttttgcatt ttgcggaagg catgagaaat gccaaaacaa gtaaatatag agcaaagtat 4980

tttacaacga ttgttagtaa gtatttttga ggtagatggt tggtattata ttaccaagtt 5040

tcaaagtatt cttttaccag agaacatata agtaacatat gtatgattcg aagtgcccac 5100

atttgaattt actttcgatg aaggattgat acggattttt tttccttgaa aaatgtgtaa 5160

aagcacatgt ttacaaatca tcaccatatt aagatgaaag taggcatatg gtttaaaaag 5220

ttaaaggagc tcatcaggtt taatttgttt tatgcttaca cacgtattga ggatacaatt 5280

ttaagggttg agccgttagc tcttatgcca aaaatatctc caaagaaaaa aaattgatca 5340

caattgcttg gataattgta tgagtatatc taattgaatt tgggccccat caagatgatt 5400

accatacaca ttcaactgta catggattga tatgccaaat tccggtaagt ggaggtgcca 5460

agaggaagga ggaaggattt atgctttgat ctagaaacat caaggcggca cactttcccc 5520

tttcctatat actgaggaac tcttccaggt aatacgaacc cttagctact ttcctttcat 5580

gctcaatttt cacccttctt gtgattgctt cctcaatatg ctgggaaaca agttagtagt 5640

gattattggt gccacgggaa ctggaaaaac aagactctca attgagatag ccaaggcgat 5700

tggtggggaa gtggtaaatg ctgacaagat gcaaatttac gatggcctgg atattacgac 5760

aaacaaggtt tctttacaag atcgatgcgg catacctcat caccttattg cgtccatccc 5820

tcgcaacgca ggtgattttc ctgtgtcatt ttttcgatct gctgcaaaaa ccacaataaa 5880

ctgcattgcc agacgtggtc acacaccgat tgtggtgggt ggatctaact cacttatcca 5940

tggtctcctt gttgacaatt ttgattcgtc tattgtggat ccttttgggc aattggaggt 6000

tagctatcgg ccgacgcctc gatcgcaatg ttgttttcta tgggttcatg ttaatgaggt 6060

gattcttaat gagtatttga aacgtcgtgt tgacaacatg gttgatgctg ggttagttga 6120

ggaaattgaa gaatattttg acattatc agttaatgga catgttccat atgtgggatt 6180

agggaaggcc attggtgttc cagagctaag cgagtatttt actggacggg tgagttgtag 6240

tgatgctctt tctatgatga agaccaatac acagattctt gcacgatctc aagtcacaaa 6300

gattcatcgc atggttgatg tgtggggatg gcatgttcat gcccttgatt gtactgaaac 6360

tattctagca catcttactg gatcaaataa gtatatggag gatttggtgt ggaaacgtga 6420

tgtaagtgac cctggacttg ctgctataca agattttctg tgataatatc agaagatggg 6480

aagctagttt ctcaaacaca tcggctattg attttgtcta caataatggt ttaatcgtct 6540

ggcttgctta gtaattttac agatcatggc atagtaagtt aacttggatc attttgggtg 6600

tgtttggaag gagcaaacat caattggtgt atatgaaatt acttggaggc cttttgtacc 6660

ttaaacactt ggatgccttt tattttacat aatagttata tatagttgtt gttcataatt 6720

ttttgatgtc atcaatattc atacgtgctg atgcgattct tattgattat ctctaataga 6780

tatgatgtgg tgccaacaaa aacaacaaac atggaagtca caaatagcca tataagaaaa 6840

taatagaggg ttcccagttg ttcatgcacc aagcttaata caaataggaa ataaacatga 6900

tagtccaatg acaatggacc aagtttagag tagcaccaca cacaatgctt gttcacttac 6960

tgatacaaca taaataataa agagttaagt atgacaacac aaaaaacatc ccctgcaaca 7020

aagagcccac atagagagta tacataaagt ccaaaaacaa tgtttttgtt aaatctctgg 7080

ttgggaagta attatttgtc gttacagtcg aaattttcaa acttgaaaac ttaaccatag 7140

gaatttttgg agagcccggc ctttgaggat ggacttagaa tttggaggaa attttctaag 7200

aggttgatag aacccaaacc tcaagattca aatatttgga tcaagacttt tgggcttggg 7260

attggtgtt tgaagaaaca gcggggatttg agagtactgg cacataatcc taaatacact 7320

caaagaatca aaagatttta aacataggtt tcaaataaaa aaaatcaacc gaggcaaaac 7380

ccaaggcgtt gcaatcctac cccttattaa tagaatcttg tcctgagatt tcggccaaag 7440

aagggtagca gaatgttatt gtggctcctg ttcagtgata ggctcaatac cagggccatg 7500

ttggatagaa gacattgtgc aaaggaagat gatgatctaa catgtgttgt gtgtaatggt 7560

gagtgtagag aaactcggct tcatctcttc tctgcctacc ctagcattag atgtaggcaa 7620

cacctgggaa ttgaatggaa acataacctg gaatttttcc caacggttgt tctcgcgaga 7680

ttgaggtttg gtcggagagg ttttctagaa atatttttta tagcctcatg tatatttgga 7740

aacagagaaa gaggcttatc ttccaaaata tcctgcctat gttccagtct tggaggttgc 7800

tttttgtgaa tgaagttctt ctacatatgt gtagaatgaa ggatcctcta aaacaatctg 7860

tttttgattg gttacaaacc ttataggttt tgagttttcc ctgtaatctg taactcttgt 7920

aaatatttcc cttgttttaa tgaaccttgt tttaatgaaa atacactgct aggcaaagcc 7980

ctggcagtat ttgcagttaa aaaaataggg tccttgaaac tatacatggt ctatgtgctg 8040

accttttcct ttggtggttg cggcattcct atcccatctt ttactgagtg atacatgggc 8100

cactgtttga cccaaaattt ttgaactcaa gtgtcgactc tgaactgata ctgtctttgt 8160

tgagaatcta aagttcttct ttggtgtcag tgctggtgtt gttatgtcct gatcgggaaa 8220

taatggggac ctctatttgg atgtgttgtg gccatatcct gcatccttgc cggttgttat 8280

gacggtcatt cggggtattc gaggtcattt ctcagcctct atacatgttc accaacatac 8340

ttttttttac ctcgtgcact tgggtaccca tttcattcag cgcaccctta tcttcggggg 8400

cccgtctctg tctccttgga gtggttttgg tcgtgcacgc aggtgtggcg agtggaggcg 8460

gtg 8463

<210>43

<211>590

<212>PRT

<213> Rice

<220>

<221>CHAIN

<222>(0)...(0)

<223> OsIPT11 amino acid sequence (006475_2)

<400>43

Met Glu Asn Ser Ser Lys Lys Thr Gln Glu Phe Phe Pro Lys Gly Gly

1 5 10 15

Asn Gly Gly Tyr Ala Glu Gln Leu Glu Leu Leu Leu Lys Gln Leu Arg

20 25 30

Phe Pro Asn Lys Pro Ile His His Ala Glu Gln Val Ile Lys Gly Phe

35 40 45

Arg Lys Asp Trp Thr Met Lys Ile Tyr Ile Gln Ala Arg Glu Glu Lys

50 55 60

Cys Gln Gly His Val Phe Lys Ser Arg His Leu Arg Ala Asn Lys Glu

65 70 75 80

Ala Ala Leu Gln Asp Ala Ser Arg Glu Ala Phe Met Arg Leu Cys Lys

85 90 95

Ile Tyr Ser Ile Glu Val Ala Ser Thr Pro Phe Phe Leu His Pro Phe

100 105 110

Arg Glu Cys Gly Asp Arg Arg Cys His Ile Arg Lys Phe Arg Gly Phe

115 120 125

Glu Glu Gln Ser Pro Ile His Phe Ser Met Trp Met Trp Ala Ala Asp

130 135 140

Glu Ala Tyr Glu Glu Ala Leu Glu Glu Leu Asp Met Leu Arg Ser Lys

145 150 155 160

Ile Ala Gly Trp Glu Glu Arg Tyr Asn His Leu Ala Lys Glu His Thr

165 170 175

Thr Arg Gly Gln Leu Leu Glu Ala Ile Lys Leu Arg Leu Gln Trp Tyr

180 185 190

Phe Arg Thr Pro Ser Gln Ala His Ile Gln Arg Thr Leu Pro Pro Pro

195 200 205

Pro Gln Arg Val Thr Arg Ser Asp Gly Glu Asp Tyr Ser Gln Ile Asn

210 215 220

Ala His Gln Ala Cys Leu Glu Arg Ser Glu Val Lys Leu Asp Arg Ala

225 230 235 240

Thr Ser Gln Asp Tyr Leu Gln Gly Tyr Lys Pro Pro Ser Glu Ser Leu

245 250 255

Asp Ala Ile Val Trp Pro Leu Val Glu Gly Lys His Asp Asn Thr Ser

260 265 270

Ser Gly Arg Arg Asn Glu Lys Ala Trp Glu Met Ala Lys Gln Val Ile

275 280 285

Arg Thr Leu Ser Tyr Phe Pro Phe Met Leu Asn Phe His Pro Ser Cys

290 295 300

Asp Cys Phe Leu Asn Met Leu Gly Asn Lys Leu Val Val Ile Ile Gly

305 310 315 320

Ala Thr Gly Thr Gly Lys Thr Arg Leu Ser Ile Glu Ile Ala Lys Ala

325 330 335

Ile Gly Gly Glu Val Val Asn Ala Asp Lys Met Gln Ile Tyr Asp Gly

340 345 350

Leu Asp Ile Thr Thr Asn Lys Val Ser Leu Gln Asp Arg Cys Gly Ile

355 360 365

Pro His His Leu Ile Ala Ser Ile Pro Arg Asn Ala Gly Asp Phe Pro

370 375 380

Val Ser Phe Phe Arg Ser Ala Ala Lys Thr Thr Ile Asn Cys Ile Ala

385 390 395 400

Arg Arg Gly His Thr Pro Ile Val Val Gly Gly Ser Asn Ser Leu Ile

405 410 415

His Gly Leu Leu Val Asp Asn Phe Asp Ser Ser Ile Val Asp Pro Phe

420 425 430

Gly Gln Leu Glu Val Ser Tyr Arg Pro Thr Pro Arg Ser Gln Cys Cys

435 440 445

Phe Leu Trp Val His Val Asn Glu Val Ile Leu Asn Glu Tyr Leu Lys

450 455 460

Arg Arg Val Asp Asn Met Val Asp Ala Gly Leu Val Glu Glu Ile Glu

465 470 475 480

Glu Tyr Phe Asp Thr Leu Ser Val Asn Gly His Val Pro Tyr Val Gly

485 490 495

Leu Gly Lys Ala Ile Gly Val Pro Glu Leu Ser Glu Tyr Phe Thr Gly

500 505 510

Arg Val Ser Cys Ser Asp Ala Leu Ser Met Met Lys Thr Asn Thr Gln

515 520 525

Ile Leu Ala Arg Ser Gln Val Thr Lys Ile His Arg Met Val Asp Val

530 535 540

Trp Gly Trp His Val His Ala Leu Asp Cys Thr Glu Thr Ile Leu Ala

545 550 555 560

His Leu Thr Gly Ser Asn Lys Tyr Met Glu Asp Leu Val Trp Lys Arg

565 570 575

Asp Val Ser Asp Pro Gly Leu Ala Ala Ile Gln Asp Phe Leu

580 585 590

<210>44

<211>4470

<212>DNA

<213> Rice

<220>

<221> CDS

<222>(1484)...(2470)

<221>misc_feature

<222>(0)...(0)

<223> OsIPT2 full-length sequence (0188301)

<221>misc_feature

<222>2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2885,

2886, 2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895,

2896, 2897, 2898, 2899, 2900, 2901, 2902, 2903, 2904, 2905,

2906, 2907, 2908, 2909, 2910, 2911, 2912, 2913, 2914

<223>n=A, T, C or G

<221>misc_feature

<222> 2915, 2916, 2917, 2918, 2919, 2920, 2921, 2922, 2923, 2924,

2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932, 2933, 2934,

2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944,

2945, 2946, 2947, 2948, 2949, 2950, 2951, 2952, 2953

<223>n=A, T, C or G

<221>misc_feature

<222> 2954, 2955, 2956, 2957, 2958, 2959, 2960, 2961, 2962, 2963,

2964, 2965, 2966, 2967, 2968, 2969, 2970, 2971, 2972, 2973,

2974, 2975

<223>n=A, T, C or G

<400>44

aaaacaatca cacactgtta tgagctctgt tattatgccc gcaataattt ctaatctata 60

ggctacaagt aaaaaaaaag agcagaggta agtactagac tccatgcatt ctgtatattt 120

actttactct ctaagataga gttccactaa attatggatt tcaggatagg gatatcagta 180

ctacatattt tcaggcataa ataaagaata aaccatgctc aacttataca aacttgcgtg 240

atagatgtaa atagatacaa cttaccaatc gttcgtcccg atgcatcaac tttcctagtc 300

cattcaaacc caggctaaaa cattccatgc aaacagaaat aatctatgcg gtacacatgt 360

aagtttaggt attgaaccac atgcttgtgt tatgcactac tatagcaaat taactgaact 420

agagcaaata ggaatacaaa atccctcaga tgcaactgag ttttggcatt gaatcagtac 480

agaagctact gcttgatttt aattctttag tgcttgaacc atcataaaat agccgcaaag 540

attaaaaaaa aaacaacaac acaatatata gagaccatca tagtaatctg atccttccac 600

tttcactttt gtacgaagct gctgcatttc tgctgtctaa tcaacattct ataaacaaca 660

tcataatgtt gtctcattac acaactgtaa cctagagtat cagccagctt gggctaggat 720

atagacctaa atttcatcaa tgagtgccac atcgaatcat tttcacttac gtgcattgtt 780

ttggcccgag tttgcacgag ataagcaagt cgttctatct acttcacgta acatgtgagt 840

ttgtgcaccg cgagtgcaat ttactcaaaa taaattgcat catcatcttt ttgtgatact 900

ccgtggtttc gaaactatta agattcagat agttgtattg catagtttca aatataaaca 960

aatcacattt ttttttgtgtgtgtgtgtgtgt gggggggggg ggggttaaat tatggtgttc 1020

catagtttca cttgacaatt tcactttaaa ttcaaattta aaatttggag cctttaagtt 1080

tgtgaacaaa agtacgagat tggtcctcca caaattgaat catgtgcatg aagttgtcac 1140

aggctcacag cgactgcaca acagcagctg gaataacaca aaaaaggcca tttttatcac 1200

tatgccattc atatgtatta aattatctct actctttctg ttcgagtgat ttgaacattt 1260

tcacatccag gtataatcca tgctaacacc aggacgtgtt ctcatttcag ctataaatag 1320

caaaaaaaat tcaaatatgt aaaacccgt caccgttctc atccaaaatt atctactttc 1380

ccgataattt cattttcatt aactccattc ccgatcagtg agattttgct acgcattgtt 1440

attgatataa aaagatggct ataccttgga tgcgagtgtg gcc atg gag cac tgc 1495

                        

1

aat ggc atc gcc gcc gtt ggg cgc tgg ttg tcc acc aag ccc aag gtt 1543

Asn Gly Ile Ala Ala Val Gly Arg Trp Leu Ser Thr Lys Pro Lys Val

5 10 15 20

atc ttc gtg ctc ggc gcc acc gcc acc ggc aag tcc aag ctc gcc atc 1591

Ile Phe Val Leu Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu Ala Ile

25 30 35

cgc ctc gcc gcg cgc ttc gac ggc gag gtc atc aac tcc gac aag atc 1639

Arg Leu Ala Ala Arg Phe Asp Gly Glu Val Ile Asn Ser Asp Lys Ile

40 45 50

cag gcg cac gac ggc ttc ccg gtc atc acc aac aag gtc acc gac gag 1687

Gln Ala His Asp Gly Phe Pro Val Ile Thr Asn Lys Val Thr Asp Glu

55 60 65

gag cgt gcc ggc gtc gcg cac cac ctc ctc ggc ggc gtc agc ccc gac 1735

Glu Arg Ala Gly Val Ala His His Leu Leu Gly Gly Val Ser Pro Asp

70 75 80

gcc gac ttc acc gcg gag gac ttc cgc cgc gag gcg gcc gcc gcc gtc 1783

Ala Asp Phe Thr Ala Glu Asp Phe Arg Arg Glu Ala Ala Ala Ala Val

85 90 95 100

gcc cgc gtc cac gcg gcc ggc cgc ctc ccc gtc gtc gcc ggc ggg tcg 1831

Ala Arg Val His Ala Ala Gly Arg Leu Pro Val Val Ala Gly Gly Ser

105 110 115

aac atc tac gtc gag gcg ctc gtg gcc ggc ggc ggc ggc gcg ttc ctc 1879

Asn Ile Tyr Val Glu Ala Leu Val Ala Gly Gly Gly Gly Ala Phe Leu

120 125 130

gcg gcg tac gac tgc ctc ttc ctg tgg acc gac gtc gcg ccg gac ctg 1927

Ala Ala Tyr Asp Cys Leu Phe Leu Trp Thr Asp Val Ala Pro Asp Leu

135 140 145

ctg cgg tgg tac acg gcg gcg cgc gtg gac gac atg gtg cgg cgc ggg 1975

Leu Arg Trp Tyr Thr Ala Ala Arg Val Asp Asp Met Val Arg Arg Gly

150 155 160

ctg gtt ggc gag gcc cgc gcc ggg ttc gac gcc ggg gcg gac tac acc 2023

Leu Val Gly Glu Ala Arg Ala Gly Phe Asp Ala Gly Ala Asp Tyr Thr

165 170 175 180

cgc ggc gtg cgc cgc gcc atc ggg cta ccc gag atg cac ggc tac ctg 2071

Arg Gly Val Arg Arg Ala Ile Gly Leu Pro Glu Met His Gly Tyr Leu

185 190 195

ctg gcg gag cgc gag ggc ggc gcc ggc gcc gag gac gac gac gac ctc 2119

Leu Ala Glu Arg Glu Gly Gly Ala Gly Ala Glu Asp Asp Asp Asp Leu

200 205 210

ctc gcc ggc atg ctc gag gcc gcc gtg cgc gag atc aag gac aac acg 2167

Leu Ala Gly Met Leu Glu Ala Ala Val Arg Glu Ile Lys Asp Asn Thr

215 220 225

ttc cgc ctc acc gtg tcg cag gtg gcc aag atc cgg cgc ctc agc gcg 2215

Phe Arg Leu Thr Val Ser Gln Val Ala Lys Ile Arg Arg Leu Ser Ala

230 235 240

ctg ccc ggg tgg gac gtc cgg cgc gtg gac gcg acg gcg gtg gtg gcg 2263

Leu Pro Gly Trp Asp Val Arg Arg Val Asp Ala Thr Ala Val Val Ala

245 250 255 260

cgc atg gcg gag ggc gcg ccc cac ggc gag acg tgg agg gag gtg gtg 2311

Arg Met Ala Glu Gly Ala Pro His Gly Glu Thr Trp Arg Glu Val Val

265 270 275

tgg gag ccg tgc gag gag atg gtc agc cgcttc ctc gag acg ccc gcc 2359

Trp Glu Pro Cys Glu Glu Met Val Ser Arg Phe Leu Glu Thr Pro Ala

280 285 290

gcc gcc gcc gcc gtc gtt gcc aac ggc aaa gtc gac gtg aac gtc ggc 2407

Ala Ala Ala Ala Val Val Ala Asn Gly Lys Val Asp Val Asn Val Gly

295 300 305

gac gcg gcc gcc ggc ttg cct gag gct gcc gcc gcc gcc gtc gtt gcg 2455

Asp Ala Ala Ala Gly Leu Pro Glu Ala Ala Ala Ala Ala Val Val Ala

310 315 320

gcg ggt gtg gtc taa ctctaagtag gatacgcggc gacggtgcat gtttgcatgg 2510

Ala Gly Val Val *

325

tgggtggcgg ctcatgttgc ggttttgggt tggctttggc gtggctgggc caggtggctt 2570

gcaatatttc attatttatt tatttatttt tattttgagc tgcagcgata tgagattttg 2630

agtgagaaag gagagggagg gagacacaag tatctttgag cttgtttaag cttagtgtta 2690

caagagatta ttttgttatg ttttcagaat atataaaatg ctagcgcctc tagtataatc 2750

ggtagtattt gacaccgcac aaaatagtag agaatgctac gcagcgtcaa atattaccag 2810

ttgagggaat acaaattcta aagtgtttac ttgtcttttg atttgaagtt taaaaccatc 2870

gaaatnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2930

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnncgtgc gaggagatgg 2990

tcagccgctt cctcgagacg cccgccgccg ccgccgccgt cgttgccaac ggcaaagtcg 3050

acgtgaacgt cggcgacgcg gccgccggcg tgcctgaggc tgccgccgcc gccgtcgttg 3110

cggcgggtgt ggtctaactc taagtaggat acgcggcgac ggtgcatgtt tgcatggtgg 3170

gtggcggctc atgttgcggt tttgggttgg ctttggcgtg gctgggccag gtggcttgca 3230

atatttcatt atttatttttttattttg agctgcagtg atatgagatc ttgagtgaga 3290

aaggagaggt agggagaacac aagtatcttt gagcttgttt aagcttagtg ttacaagaga 3350

ttattttgtt atgttttcag aatatataaa atgctagcgc ctctagtata atcggtagta 3410

tttgacaccg cacaaaatag tagagaatgc tacgcagcgt caaatattac cagttgaggg 3470

aatacaaatt ctaaagtgtt tacttgtctt ttgatttgaa gtttaaaacc aatcgaaatt 3530

cttaactgtc ttttgaaatt cgaagtgttt tctcccttat tagggcctgt tcggaacaaa 3590

aggataaaaa acacagaaat atgatagagc gtaaattgga aaactcaggg actgtaaaac 3650

ttgagctgtt tggaacagag gaatgctagg ggatagatac acaagcacac aactaatgtg 3710

aaggaaaatt tcctttagga ggaaccttat ttttctttgt ttccttttaa agtatatgat 3770

ttcaaaaaac aatatttgga gggagagatg tccctccaaa taatttttaa gaaaaaaata 3830

tgagcgtgtt ggagattaaa cacggagttc aaacaaacaa gatttggagg gagagacgtc 3890

cctccaaaca ttttttaaga aaaaattatg agcgtgttgg ggattaaaca cggaacctca 3950

gggttgaaat catatatctc ttgtcactgc actatcaagt acatctcaaa gtacagagtt 4010

tctcaatgct cttttctttg atccaaacaa catcatagga agattttcca aaggaaacta 4070

aacctccaca attcctttaa aattcctttg atccaaccat gccttggtta ctgcatattt 4130

gttttagtat aaccttatat tgcttgaaac taaacttccc ttcttttcat actacctgac 4190

agattgttag ttctaagagt atgcttatct aacatacgga ttataagcca tagacaattt 4250

taaaatttcg atcttaattt tcgaattaat ttagttttat ttcttatttt cagccttagt 4310

ttttgaaatg ctaagactag aagtataaat ttcttactag ttgctttggt cacgcgttgt 4370

tggcttataa accacagcac acaagaggaa attatttatt tgtatttaca aatctgtacc 4430

ttcaagtatt cttagtttac cgcacggtgg caaagaaatg 4470

<210>45

<211>984

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT2 coding sequence (018830_1)

<221> CDS

<222>(1)...(984)

<400>45

atg gag cac tgc aat ggc atc gcc gcc gtt ggg cgc tgg ttg tcc acc 48

Met Glu His Cys Asn Gly Ile Ala Ala Val Gly Arg Trp Leu Ser Thr

1 5 10 15

aag ccc aag gtt atc ttc gtg ctc ggc gcc acc gcc acc ggc aag tcc 96

Lys Pro Lys Val Ile Phe Val Leu Gly Ala Thr Ala Thr Gly Lys Ser

20 25 30

aag ctc gcc atc cgc ctc gcc gcg cgc ttc gac ggc gag gtc atc aac 144

Lys Leu Ala Ile Arg Leu Ala Ala Arg Phe Asp Gly Glu Val Ile Asn

35 40 45

tcc gac aag atc cag gcg cac gac ggc ttc ccg gtc atc acc aac aag 192

Ser Asp Lys Ile Gln Ala His Asp Gly Phe Pro Val Ile Thr Asn Lys

50 55 60

gtc acc gac gag gag cgt gcc ggc gtc gcg cac cac ctc ctc ggc ggc 240

Val Thr Asp Glu Glu Arg Ala Gly Val Ala His His Leu Leu Gly Gly

65 70 75 80

gtc agc ccc gac gcc gac ttc acc gcg gag gac ttc cgc cgc gag gcg 288

Val Ser Pro Asp Ala Asp Phe Thr Ala Glu Asp Phe Arg Arg Glu Ala

85 90 95

gcc gcc gcc gtc gcc cgc gtc cac gcg gcc ggc cgc ctc ccc gtc gtc 336

Ala Ala Ala Val Ala Arg Val His Ala Ala Gly Arg Leu Pro Val Val

100 105 110

gcc ggc ggg tcg aac atc tac gtc gag gcg ctc gtg gcc ggc ggc ggc 384

Ala Gly Gly Ser Asn Ile Tyr Val Glu Ala Leu Val Ala Gly Gly Gly

115 120 125

ggc gcg ttc ctc gcg gcg tac gac tgc ctc ttc ctg tgg acc gac gtc 432

Gly Ala Phe Leu Ala Ala Tyr Asp Cys Leu Phe Leu Trp Thr Asp Val

130 135 140

gcg ccg gac ctg ctg cgg tgg tac acg gcg gcg cgc gtg gac gac atg 480

Ala Pro Asp Leu Leu Arg Trp Tyr Thr Ala Ala Arg Val Asp Asp Met

145 150 155 160

gtg cgg cgc ggg ctg gtt ggc gag gcc cgc gcc ggg ttc gac gcc ggg 528

Val Arg Arg Gly Leu Val Gly Glu Ala Arg Ala Gly Phe Asp Ala Gly

165 170 175

gcg gac tac acc cgc ggc gtg cgc cgc gcc atc ggg cta ccc gag atg 576

Ala Asp Tyr Thr Arg Gly Val Arg Arg Ala Ile Gly Leu Pro Glu Met

180 185 190

cac ggc tac ctg ctg gcg gag cgc gag ggc ggc gcc ggc gcc gag gac 624

His Gly Tyr Leu Leu Ala Glu Arg Glu Gly Gly Ala Gly Ala Glu Asp

195 200 205

gac gac gac ctc ctc gcc ggc atg ctc gag gcc gcc gtg cgc gag atc 672

Asp Asp Asp Leu Leu Ala Gly Met Leu Glu Ala Ala Val Arg Glu Ile

210 215 220

aag gac aac acg ttc cgc ctc acc gtg tcg cag gtg gcc aag atc cgg 720

Lys Asp Asn Thr Phe Arg Leu Thr Val Ser Gln Val Ala Lys Ile Arg

225 230 235 240

cgc ctc agc gcg ctg ccc ggg tgg gac gtc cgg cgc gtg gac gcg acg 768

Arg Leu Ser Ala Leu Pro Gly Trp Asp Val Arg Arg Val Asp Ala Thr

245 250 255

gcg gtg gtg gcg cgc atg gcg gag ggc gcg ccc cac ggc gag acg tgg 816

Ala Val Val Ala Arg Met Ala Glu Gly Ala Pro His Gly Glu Thr Trp

260 265 270

agg gag gtg gtg tgg gag ccg tgc gag gag atg gtc agc cgc ttc ctc 864

Arg Glu Val Val Trp Glu Pro Cys Glu Glu Met Val Ser Arg Phe Leu

275 280 285

gag acg ccc gcc gcc gcc gcc gcc gtc gtt gcc aac ggc aaa gtc gac 912

Glu Thr Pro Ala Ala Ala Ala Ala Val Val Ala Asn Gly Lys Val Asp

290 295 300

gtg aac gtc ggc gac gcg gcc gcc ggc ttg cct gag gct gcc gcc gcc 960

Val Asn Val Gly Asp Ala Ala Ala Gly Leu Pro Glu Ala Ala Ala Ala

305 310 315 320

gcc gtc gtt gcg gcg ggt gtg gtc 984

Ala Val Val Ala Ala Gly Val Val

325

<210>46

<211>328

<212>PRT

<213> Rice

<400>46

Met Glu His Cys Asn Gly Ile Ala Ala Val Gly Arg Trp Leu Ser Thr

1 5 10 15

Lys Pro Lys Val Ile Phe Val Leu Gly Ala Thr Ala Thr Gly Lys Ser

20 25 30

Lys Leu Ala Ile Arg Leu Ala Ala Arg Phe Asp Gly Glu Val Ile Asn

35 40 45

Ser Asp Lys Ile Gln Ala His Asp Gly Phe Pro Val Ile Thr Asn Lys

50 55 60

Val Thr Asp Glu Glu Arg Ala Gly Val Ala His His Leu Leu Gly Gly

65 70 75 80

Val Ser Pro Asp Ala Asp Phe Thr Ala Glu Asp Phe Arg Arg Glu Ala

85 90 95

Ala Ala Ala Val Ala Arg Val His Ala Ala Gly Arg Leu Pro Val Val

100 105 110

Ala Gly Gly Ser Asn Ile Tyr Val Glu Ala Leu Val Ala Gly Gly Gly

115 120 125

Gly Ala Phe Leu Ala Ala Tyr Asp Cys Leu Phe Leu Trp Thr Asp Val

130 135 140

Ala Pro Asp Leu Leu Arg Trp Tyr Thr Ala Ala Arg Val Asp Asp Met

145 150 155 160

Val Arg Arg Gly Leu Val Gly Glu Ala Arg Ala Gly Phe Asp Ala Gly

165 170 175

Ala Asp Tyr Thr Arg Gly Val Arg Arg Ala Ile Gly Leu Pro Glu Met

180 185 190

His Gly Tyr Leu Leu Ala Glu Arg Glu Gly Gly Ala Gly Ala Glu Asp

195 200 205

Asp Asp Asp Leu Leu Ala Gly Met Leu Glu Ala Ala Val Arg Glu Ile

210 215 220

Lys Asp Asn Thr Phe Arg Leu Thr Val Ser Gln Val Ala Lys Ile Arg

225 230 235 240

Arg Leu Ser Ala Leu Pro Gly Trp Asp Val Arg Arg Val Asp Ala Thr

245 250 255

Ala Val Val Ala Arg Met Ala Glu Gly Ala Pro His Gly Glu Thr Trp

260 265 270

Arg Glu Val Val Trp Glu Pro Cys Glu Glu Met Val Ser Arg Phe Leu

275 280 285

Glu Thr Pro Ala Ala Ala Ala Ala Val Val Ala Asn Gly Lys Val Asp

290 295 300

Val Asn Val Gly Asp Ala Ala Ala Gly Leu Pro Glu Ala Ala Ala Ala

305 310 315 320

Ala Val Val Ala Ala Gly Val Val

325

<210>47

<211>4114

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT1 genome sequence (006704_3)

<221> CDS

<222>(2001)...(2978)

<400>47

tttgcttgca aggttttgtg tcgcaatgct agtttctttt tcttttttct tgttaatatc 60

catttttctt tttcttttat caaaaaaaaa tatgtgggtg taggggtgga tctaggggga 120

aaaaagcagg ggctaaaaaa actagacaaa gctccaactc attttcggcc tttctgtatc 180

tttatcggaa aattttggag gctcaagggt ctctaataat ccatatatta gtagcgggcg 240

ctcgaacctc gtctccgtga tagatccata tatatgggtg gtttttgttt gatctaaaaa 300

gatttttttt tctttgtaat ccttatgttg agcgatagat attttatttt tacgttagat 360

atattagata tctaagttga ggcttttttt aagggctaat atttttcatt cacatagatc 420

tcaatgttat tataattttt ccatgtcata tataaactta agaccgaact aatgcatctt 480

gtttttacct ttgactttta attacctttc aataggacct aaaagggatt agggagtcct 540

aattgcccta ggctttgtct ttacccccttg tatgatgtga acaccagaca taggttcaat 600

aggaatgaat agtttgcgga tacatatgag ataatgtttt gcgctatatg tcaaagttag 660

aaggtggtga acataaaaca aagttaaagt ttcactgaat aataactagc gacgatgaac 720

ataaagaaat tattgaagtt taccctcaag aagtagtaat attaatgatt gattttgtta 780

attttgctag ctagaatatg catacagcag cagatgttat catccttttt atttatacgt 840

actgtcatcc ttttctcctt tggtatagat tggttttatg aagttgatgg aaaattgacc 900

acagatgagt ttaattattca ttcatgaacc gtgaacttct cttaaaaaaa gtaaaataag 960

gaagttcaat gcgaataatc tatatggggg attaattact tgtcaatgct tttcatgata 1020

tttaatgttt tcgactgtaa gattaacaaa ttgtgcagag atgtattttt catgcatgat 1080

aatttggaag tgcacccaca ttgttccatg aacttgcatt gctagtttta tgttaaattc 1140

tcgttcacaa caaattaact agagacaatt aattatcatg atatataggt aatttagcat 1200

caaactgatc atttacaatc tgcattactg tttttgaaaa caaaaataat aaaaaaatgc 1260

tatatcgtta caacggtggt ttattttact aacatcatca tgacatcaac atgggatcta 1320

ggtatcaata ctgttaccta tgggtatcat gtatgatacc ccgatattag atatggttcc 1380

tatgtttatc atgtctaatg ctagtatcat ataagtacca aatttgtgcc atgctggcgt 1440

tatggtgatg ccagaaataa agtattccat tctggaatgg tatagtgtct tctggtaatc 1500

cttatttaga aaatgtatta taaccattta aatatagtat atgttcccct tccatctcaa 1560

aatataaatg attttgggta gatgtaatct atagtcctta aaatgttagt agttctggtg 1620

gtgtccattc ccacaccatc tctactacta ccgtgtgagc cccacaatct atactattac 1680

attatctttt aacgagtcct cccccgtttc accgttggct cttagtactt ggactaaata 1740

agtttttgtt aattgattta ataaattaga aactcaattt gttgtccgtt acaaagcaca 1800

agctcttagc tatcctaata ttattaatct gaacaaactt atatcacatc catctaaaat 1860

ctcgtgtatt atattttggg acgggggagt aagaatgatc gagtgcacgt aattttagg 1920

tcgtaaattt actatctttg caaaaagtaa ttaacgatgg cttatatacc tcttctccgg 1980

tgagcagctc accttcatca atg gag tac cac gtc ggc ggt gtc atc gga cag 2033

                                      Met Glu Tyr His Val Gly Gly Val Ile Gly Gln

1 5 10

tca ccc aag ccc aag gtc gtc ttc gtg ctc ggc gcc acc gcc acc ggc 2081

Ser Pro Lys Pro Lys Val Val Phe Val Leu Gly Ala Thr Ala Thr Gly

15 20 25

aag tcc aag ctc gcc atc tcc atc gcc gag cgg ttc ggc ggc gag gtg 2129

Lys Ser Lys Leu Ala Ile Ser Ile Ala Glu Arg Phe Gly Gly Glu Val

30 35 40

atc aac tcc gac aag atc cag gtg cac gac ggg ttc ccc atc atc acg 2177

Ile Asn Ser Asp Lys Ile Gln Val His Asp Gly Phe Pro Ile Ile Thr

45 50 55

aac aag gtc acc gag gag gag cgc gcc ggc gtc ccc cac cac ctc ctc 2225

Asn Lys Val Thr Glu Glu Glu Arg Ala Gly Val Pro His His Leu Leu

60 65 70 75

ggc gtc ctc cac ccg gac gcc gac ttc acc gcc gag gac ttc cgg cgc 2273

Gly Val Leu His Pro Asp Ala Asp Phe Thr Ala Glu Asp Phe Arg Arg

80 85 90

gag gcg gcc gcc gcc gtc gcc cgc gtc ctc gcg gcg ggc cgc ctc ccc 2321

Glu Ala Ala Ala Ala Val Ala Arg Val Leu Ala Ala Gly Arg Leu Pro

95 100 105

gtc gtg gcc ggc ggg tcg aac acc tac gtc gag gcg ctg gtg gag ggc 2369

Val Val Ala Gly Gly Ser Asn Thr Tyr Val Glu Ala Leu Val Glu Gly

110 115 120

ggc ggc ggc gcg ttc cgc gcg gcg cac gac tgc ctc ttc ctg tgg acc 2417

Gly Gly Gly Ala Phe Arg Ala Ala His Asp Cys Leu Phe Leu Trp Thr

125 130 135

gac gtc gcg ccg ggc ctg ctg cgg tgg tac acc gcg gcg cgc gtg gac 2465

Asp Val Ala Pro Gly Leu Leu Arg Trp Tyr Thr Ala Ala Arg Val Asp

140 145 150 155

gac atg gtg cgg cgc ggg ctg gtg ggc gag gcg cgc gcc ggg ttc gtc 2513

Asp Met Val Arg Arg Gly Leu Val Gly Glu Ala Arg Ala Gly Phe Val

160 165 170

gac ggc gcc ggc gcc gcg gac tac tac acc cgc ggc gtg cgc cgc gcc 2561

Asp Gly Ala Gly Ala Ala Asp Tyr Tyr Thr Arg Gly Val Arg Arg Ala

175 180 185

atc ggg atc ccg gag atg cac ggg tac ctc ctg gcc gag cgc tcg ggc 2609

Ile Gly Ile Pro Glu Met His Gly Tyr Leu Leu Ala Glu Arg Ser Gly

190 195 200

ggc gag gcg gcc gac gac ggc gag ctc gcc gcc atg ctc gac ggc gcc 2657

Gly Glu Ala Ala Asp Asp Gly Glu Leu Ala Ala Met Leu Asp Gly Ala

205 210 215

gtg cgc gag atc aag gcc aac acc tac cgc ctc gcc gcg acg cag gtg 2705

Val Arg Glu Ile Lys Ala Asn Thr Tyr Arg Leu Ala Ala Thr Gln Val

220 225 230 235

gcg aag atc cgg cgg ctg agc gcg ctg gac ggg tgg gac gtg cgg cgc 2753

Ala Lys Ile Arg Arg Leu Ser Ala Leu Asp Gly Trp Asp Val Arg Arg

240 245 250

gtg gac gcg acg gtg gtg gtg gcg cgc atg gcg gag ggg gcg ccg cac 2801

Val Asp Ala Thr Val Val Val Ala Arg Met Ala Glu Gly Ala Pro His

255 260 265

agg gag acg tgg gag gcg gtg gtg tgg aag ccg tgc gag gag atg gtc 2849

Arg Glu Thr Trp Glu Ala Val Val Trp Lys Pro Cys Glu Glu Met Val

270 275 280

ggc cgc ttc ctc gag gcg tcc gcc gcc gtg gat gac gac gac aat gcc 2897

Gly Arg Phe Leu Glu Ala Ser Ala Ala Val Asp Asp Asp Asp Asn Ala

285 290 295

gcc tcc ggt tcg ccg gcg gcg ttg gca ccg atg acg gcg gcg tgt cgc 2945

Ala Ser Gly Ser Pro Ala Ala Leu Ala Pro Met Thr Ala Ala Cys Arg

300 305 310 315

ctg agg gcg cag ctg gtg cag ctg caa tac taa ttagagtgga gtggcttggc 2998

Leu Arg Ala Gln Leu Val Gln Leu Gln Tyr *

320 325

gttggctagc gttagtgcta ccactataat taatatatat atagtgcaag cagatcgcgt 3058

ttgattatagag tgacaattat atgtcgtcga aagtacgttt tgtgatggaa gtaacatcat 3118

ccagtcgcta taagtgggac ccatatgtca tcagtttaat taaagagatg attttttttg 3178

cggggaaatt aaagagatga ttaggaagaa ggttttcctt atttattcac agtggagtgc 3238

tattttacaa atagttctag tatataaata tagggagatg gattttcata attggagagg 3298

acatgcattc cctcgttttt ttataccacc tatatatata gaaaaaaatg ataatataat 3358

taatacaaaa tatatcactt cacagcctcc atgttcaaat ttgtaaacaa aacaaattaa 3418

accgatacta attaatgtat gttcatagtt atatatattt gtatatttca tggtaatgat 3478

ggttttcttg cattttatat tataagatgt tttgattttt tttaaacttt taaaatttaa 3538

ttgattttat aggaaagcgt agcaacattt aatataacgc caaattaatt ttactaaatc 3598

cgacatctat agaaatgtta ctacatgcct ttatttttat aaatttgttt gaatttaaaa 3658

caatttgatt gaaaaaaaat tcaaacattg tataatataa aacaaaggaa gtatataatt 37l8

aaggtattgt agttcgttgt tgcacgtgga atctcgtgtc tggaactcga tgatctttag 3778

ttccaagaga ggaagcaaag gtagcggacg gcagggttga tcgatggcga gtggcgagat 3838

ccaatgccga ctgcaaccgt gcaactaacc gccggcgccg ccgtctgatt cgctgcgaca 3898

agctgggctg ctgggcagca gctaagcaac cgagatcatc gagacggtct cagaatctga 3958

ttctccggac cggaccagac tcggattgga caaccagtag tttaatcgat cgatcgcctg 4018

tatatatatt ttgggttagt ttatcccgta cacatagaat cgattgatcg atcggcacta 4078

ctatacagcg agcgagagag attgatcggt cgagag 4114

<210>48

<211>975

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT1 coding sequence (006704_3)

<221> CDS

<222>(1)...(975)

<400>48

atg gag tac cac gtc ggc ggt gtc atc gga cag tca ccc aag ccc aag 48

Met Glu Tyr His Val Gly Gly Val Ile Gly Gln Ser Pro Lys Pro Lys

1 5 10 15

gtc gtc ttc gtg ctc ggc gcc acc gcc acc ggc aag tcc aag ctc gcc 96

Val Val Phe Val Leu Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu Ala

20 25 30

atc tcc atc gcc gag cgg ttc ggc ggc gag gtg atc aac tcc gac aag 144

Ile Ser Ile Ala Glu Arg Phe Gly Gly Glu Val Ile Asn Ser Asp Lys

35 40 45

atc cag gtg cac gac ggg ttc ccc atc atc acg aac aag gtc acc gag 192

Ile Gln Val His Asp Gly Phe Pro Ile Ile Thr Asn Lys Val Thr Glu

50 55 60

gag gag cgc gcc ggc gtc ccc cac cac ctc ctc ggc gtc ctc cac ccg 240

Glu Glu Arg Ala Gly Val Pro His His Leu Leu Gly Val Leu His Pro

65 70 75 80

gac gcc gac ttc acc gcc gag gac ttc cgg cgc gag gcg gcc gcc gcc 288

Asp Ala Asp Phe Thr Ala Glu Asp Phe Arg Arg Glu Ala Ala Ala Ala

85 90 95

gtc gcc cgc gtc ctc gcg gcg ggc cgc ctc ccc gtc gtg gcc ggc ggg 336

Val Ala Arg Val Leu Ala Ala Gly Arg Leu Pro Val Val Ala Gly Gly

100 105 110

tcg aac acc tac gtc gag gcg ctg gtg gag ggc ggc ggc ggc gcg ttc 384

Ser Asn Thr Tyr Val Glu Ala Leu Val Glu Gly Gly Gly Gly Ala Phe

115 120 125

cgc gcg gcg cac gac tgc ctc ttc ctg tgg acc gac gtc gcg ccg ggc 432

Arg Ala Ala His Asp Cys Leu Phe Leu Trp Thr Asp Val Ala Pro Gly

130 135 140

ctg ctg cgg tgg tac acc gcg gcg cgc gtg gac gac atg gtg cgg cgc 480

Leu Leu Arg Trp Tyr Thr Ala Ala Arg Val Asp Asp Met Val Arg Arg

145 150 155 160

ggg ctg gtg ggc gag gcg cgc gcc ggg ttc gtc gac ggc gcc ggc gcc 528

Gly Leu Val Gly Glu Ala Arg Ala Gly Phe Val Asp Gly Ala Gly Ala

165 170 175

gcg gac tac tac acc cgc ggc gtg cgc cgc gcc atc ggg atc ccg gag 576

Ala Asp Tyr Tyr Thr Arg Gly Val Arg Arg Ala Ile Gly Ile Pro Glu

180 185 190

atg cac ggg tac ctc ctg gcc gag cgc tcg ggc ggc gag gcg gcc gac 624

Met His Gly Tyr Leu Leu Ala Glu Arg Ser Gly Gly Glu Ala Ala Asp

195 200 205

gac ggc gag ctc gcc gcc atg ctc gac ggc gcc gtg cgc gag atc aag 672

Asp Gly Glu Leu Ala Ala Met Leu Asp Gly Ala Val Arg Glu Ile Lys

210 215 220

gcc aac acc tac cgc ctc gcc gcg acg cag gtg gcg aag atc cgg cgg 720

Ala Asn Thr Tyr Arg Leu Ala Ala Thr Gln Val Ala Lys Ile Arg Arg

225 230 235 240

ctg agc gcg ctg gac ggg tgg gac gtg cgg cgc gtg gac gcg acg gtg 768

Leu Ser Ala Leu Asp Gly Trp Asp Val Arg Arg Val Asp Ala Thr Val

245 250 255

gtg gtg gcg cgc atg gcg gag ggg gcg ccg cac agg gag acg tgg gag 816

Val Val Ala Arg Met Ala Glu Gly Ala Pro His Arg Glu Thr Trp Glu

260 265 270

gcg gtg gtg tgg aag ccg tgc gag gag atg gtc ggc cgc ttc ctc gag 864

Ala Val Val Trp Lys Pro Cys Glu Glu Met Val Gly Arg Phe Leu Glu

275 280 285

gcg tcc gcc gcc gtg gat gac gac gac aat gcc gcc tcc ggt tcg ccg 912

Ala Ser Ala Ala Val Asp Asp Asp Asp Asn Ala Ala Ser Gly Ser Pro

290 295 300

gcg gcg ttg gca ccg atg acg gcg gcg tgt cgc ctg agg gcg cag ctg 960

Ala Ala Leu Ala Pro Met Thr Ala Ala Cys Arg Leu Arg Ala Gln Leu

305 310 315 320

gtg cag ctg caa tac 975

Val Gln Leu Gln Tyr

325

<210>49

<211>325

<212>PRT

<213> Rice

<400>49

Met Glu Tyr His Val Gly Gly Val Ile Gly Gln Ser Pro Lys Pro Lys

1 5 10 15

Val Val Phe Val Leu Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu Ala

20 25 30

Ile Ser Ile Ala Glu Arg Phe Gly Gly Glu Val Ile Asn Ser Asp Lys

35 40 45

Ile Gln Val His Asp Gly Phe Pro Ile Ile Thr Asn Lys Val Thr Glu

50 55 60

Glu Glu Arg Ala Gly Val Pro His His Leu Leu Gly Val Leu His Pro

65 70 75 80

Asp Ala Asp Phe Thr Ala Glu Asp Phe Arg Arg Glu Ala Ala Ala Ala

85 90 95

Val Ala Arg Val Leu Ala Ala Gly Arg Leu Pro Val Val Ala Gly Gly

100 105 110

Ser Asn Thr Tyr Val Glu Ala Leu Val Glu Gly Gly Gly Gly Ala Phe

115 120 125

Arg Ala Ala His Asp Cys Leu Phe Leu Trp Thr Asp Val Ala Pro Gly

130 135 140

Leu Leu Arg Trp Tyr Thr Ala Ala Arg Val Asp Asp Met Val Arg Arg

145 150 155 160

Gly Leu Val Gly Glu Ala Arg Ala Gly Phe Val Asp Gly Ala Gly Ala

165 170 175

Ala Asp Tyr Tyr Thr Arg Gly Val Arg Arg Ala Ile Gly Ile Pro Glu

180 185 190

Met His Gly Tyr Leu Leu Ala Glu Arg Ser Gly Gly Glu Ala Ala Asp

195 200 205

Asp Gly Glu Leu Ala Ala Met Leu Asp Gly Ala Val Arg Glu Ile Lys

210 215 220

Ala Asn Thr Tyr Arg Leu Ala Ala Thr Gln Val Ala Lys Ile Arg Arg

225 230 235 240

Leu Ser Ala Leu Asp Gly Trp Asp Val Arg Arg Val Asp Ala Thr Val

245 250 255

Val Val Ala Arg Met Ala Glu Gly Ala Pro His Arg Glu Thr Trp Glu

260 265 270

Ala Val Val Trp Lys Pro Cys Glu Glu Met Val Gly Arg Phe Leu Glu

275 280 285

Ala Ser Ala Ala Val Asp Asp Asp Asp Asn Ala Ala Ser Gly Ser Pro

290 295 300

Ala Ala Leu Ala Pro Met Thr Ala Ala Cys Arg Leu Arg Ala Gln Leu

305 310 315 320

Val Gln Leu Gln Tyr

325

<210>50

<211>2599

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223>OsIPT5 genome sequence (027814_1)

<221> CDS

<222>(1827)...(2579)

<400>50

acctgacatc ataatacgaa caaatactgg cttgccaagt atgtggttaa caacgtgtaa 60

catagtacca atcatttagg gtggcaagac gtggcaacaa accaaacaag ccagcgagct 120

ccaggtgagt tattctgctc ttgcctcaag cccacaatcc aagttcgcac ttgatctttg 180

atctttggaa aggccatcat cgggagactg gacatgatcc aacctctcgc tttttctctt 240

cccgctacag ccaccagggc aagcagcacc gcccctgtcc aggctgattt cgcatgggtt 300

gctgatgatg gtcctcgacc tatcatccgg ctgggattat tacatgatta ttaagtaata 360

gctaattttt ttaaaaaaaa atagattaat ttgatttttt taagtaactt tcgtatagaa 420

accttttata aaaaacgtat cgtttaacag tttaaaaagc gtgcgtgcgg aaaacgagaa 480

agaggggttg ggaactcgaa ggaaagaaca cagccgggtt gggaacttgt tgctgtcacc 540

ctactccctg atgaatcctc ttgtccaggt ttttgcatgg agtaggaagt aggaactatc 600

atatcaacct agcctactag tttctcaggc atcagtagcc cagtagtgga tgcacgaacg 660

aattttacct gttgggggtg agaagctaga accgggcagg gggggaagga aggaccagca 720

acaagtggac ggatgatatg gaggctgatg gaaccgatcg tccggaggca acttgcaagc 780

aatgctacat ttgctcccgc agttgcgttg cacgacgtac tacatatgta accagaaaaa 840

tgccacgacg atgcagatcg atatatagtc aggcagcatt agcgtggtcg agtccccaac 900

gctcggcatt gcctcatcag ttaatccgca ctcgcctttt tgttagcggg acacgcatgc 960

gctgtgtgtg tgtgtgctcc cttcgatcgg gcacgagctg tgtgcggtgg catgggcgca 1020

tggcagcgac tggtcgaaac gcggccgcac gacggacacg ccgctcgatc ccccgcgcgg 1080

ctgccgtgct ccccctctcg tctggatcac cggcggcgtg ggcggctctc ttccacgagg 1140

catgagctcg gttttttttt accctctgct cgcgacggag agggaaaagg ctgctgcttc 1200

ttattcttat tccctggagc gatcagcttt ttctccgccc ctcggaggtg aaaacaaagc 1260

aacgaatgga accatggaaa cgaagtgaag gagcgtgcct tcgcaatcat ggcctgaggc 1320

cctgagcggc tgagcccctg aggcctcctg agtatatata aaccaagcat ggtttgcctc 1380

ctgcattgcc cgtgtgaatg ccaatgatac agagcccccc aagagcagag caagcgcgag 1440

aacacacacc aacaacgcaa caaaccacca ggcgtgcgcg tgcaagcgca aggcttgaaa 1500

cggagagaca cgaaaagcgc caaggtgttc gcccatcatt ataatcagct tataagggcg 1560

cgagcgaaac cgcacagttg tacacttgga ctcgcaaact agactctccg tctccttgcg 1620

ctgcgcgtta tatcggctct gcctatataa gtgtgctgag gcgactgggg ctcggtgagt 1680

gttttgggtg ggccggcttt atgagcagtc tcggtttgaa gatccgcacc gtcgtccgct 1740

cacctatggc ggccgcggcc gtcgctggcg tcggaaggga tggtagcttc gcctcccaga 1800

agcggccacg tcgggttagt gtgaga atg gag aga agc aga gtc ggg gac ggt 1853

                                                                  , 

1 5

tgc tgc tgc tcc tgc tct ggc cgc ggc ggg gtg gcg tcc act acg gcg 1901

Cys Cys Cys Ser Cys Ser Gly Arg Gly Gly Val Ala Ser Thr Thr Ala

10 15 20 25

gtc cgg ccg tcc acg ggg atg gtg gtg atc gtc ggc gcc acg ggc acg 1949

Val Arg Pro Ser Thr Gly Met Val Val Ile Val Gly Ala Thr Gly Thr

30 35 40

ggg aag acc aag ctt tcc atc gac gcg gcg cag gag ctc gcc ggc gag 1997

Gly Lys Thr Lys Leu Ser Ile Asp Ala Ala Gln Glu Leu Ala Gly Glu

45 50 55

gtg gtg aac gct gac aag att cag ctg tac gac ggc ctc gac gtc acc 2045

Val Val Asn Ala Asp Lys Ile Gln Leu Tyr Asp Gly Leu Asp Val Thr

60 65 70

acg aac aag gtg tcg ctc gcc gac cgc cgg ggc gtc ccg cac cac ctc 2093

Thr Asn Lys Val Ser Leu Ala Asp Arg Arg Gly Val Pro His His Leu

75 80 85

ctc ggc gca atc cgc gcc gag gcc ggg gag ctg ccg ccg tcg tcg ttc 2141

Leu Gly Ala Ile Arg Ala Glu Ala Gly Glu Leu Pro Pro Ser Ser Phe

90 95 100 105

cgc tcg ctc gcc gcc gcc gcc gcg gcc ggc atc gcg tcg cgc ggg cgc 2189

Arg Ser Leu Ala Ala Ala Ala Ala Ala Gly Ile Ala Ser Arg Gly Arg

110 115 120

gtg ccg gtc gtg gcc ggc ggg tcc aac tcg ctc atc cac gcg ctc ctc 2237

Val Pro Val Val Ala Gly Gly Ser Asn Ser Leu Ile His Ala Leu Leu

125 130 135

gct gac ccc atc gat gcc gcg ccg cgt gac cct ttc gcg gac gcc gat 2285

Ala Asp Pro Ile Asp Ala Ala Pro Arg Asp Pro Phe Ala Asp Ala Asp

140 145 150

gtc ggg tac cgg ccg gcg ctc cgg ttc ccg tgc tgc ctc ctc tgg gtc 2333

Val Gly Tyr Arg Pro Ala Leu Arg Phe Pro Cys Cys Leu Leu Trp Val

155 160 165

gac gtc gac gac gat gtt ctc gac gaa tac ctc gac cgg cgc gtg gac 2381

Asp Val Asp Asp Asp Val Leu Asp Glu Tyr Leu Asp Arg Arg Val Asp

170 175 180 185

gac atg gtc ggc gag ggg atg gtc gag gag ctc gag gaa tac ttc gcg 2429

Asp Met Val Gly Glu Gly Met Val Glu Glu Leu Glu Glu Tyr Phe Ala

190 195 200

acg acg tcg gcc tcg gag cgg gcc tcg cac gcc ggg ctg ggc aag gcc 2477

Thr Thr Ser Ala Ser Glu Arg Ala Ser His Ala Gly Leu Gly Lys Ala

205 210 215

atc ggc gtg ccg gag ctc ggc gac tac ttc gcc ggg cgc aag agc ctc 2525

Ile Gly Val Pro Glu Leu Gly Asp Tyr Phe Ala Gly Arg Lys Ser Leu

220 225 230

gac gcg gcg ata gac gag atc aag gcc aac acg cgg gtc ctc gcg ggc 2573

Asp Ala Ala Ile Asp Glu Ile Lys Ala Asn Thr Arg Val Leu Ala Gly

235 240 245

cgc cag gtcggcaaga tccgacgcat 2599

Arg Gln

250

<210>51

<211>773

<212>DNA

<213> Rice

<220>

<221> CDS

<222>(1)...(773)

<400>51

atg gag aga agc aga gtc ggg gac ggt tgc tgc tgc tcc tgc tct ggc 48

Met Glu Arg Ser Arg Val Gly Asp Gly Cys Cys Cys Ser Cys Ser Gly

1 5 10 15

cgc ggc ggg gtg gcg tcc act acg gcg gtc cgg ccg tcc acg ggg atg 96

Arg Gly Gly Val Ala Ser Thr Thr Ala Val Arg Pro Ser Thr Gly Met

20 25 30

gtg gtg atc gtc ggc gcc acg ggc acg ggg aag acc aag ctt tcc atc 144

Val Val Ile Val Gly Ala Thr Gly Thr Gly Lys Thr Lys Leu Ser Ile

35 40 45

gac gcg gcg cag gag ctc gcc ggc gag gtg gtg aac gct gac aag att 192

Asp Ala Ala Gln Glu Leu Ala Gly Glu Val Val Asn Ala Asp Lys Ile

50 55 60

cag ctg tac gac ggc ctc gac gtc acc acg aac aag gtg tcg ctc gcc 240

Gln Leu Tyr Asp Gly Leu Asp Val Thr Thr Asn Lys Val Ser Leu Ala

65 70 75 80

gac cgc cgg ggc gtc ccg cac cac ctc ctc ggc gca atc cgc gcc gag 288

Asp Arg Arg Gly Val Pro His His Leu Leu Gly Ala Ile Arg Ala Glu

85 90 95

gcc ggg gag ctg ccg ccg tcg tcg ttc cgc tcg ctc gcc gcc gcc gcc 336

Ala Gly Glu Leu Pro Pro Ser Ser Ser Phe Arg Ser Leu Ala Ala Ala Ala

100 105 110

gcg gcc ggc atc gcg tcg cgc ggg cgc gtg ccg gtc gtg gcc ggc ggg 384

Ala Ala Gly Ile Ala Ser Arg Gly Arg Val Pro Val Val Ala Gly Gly

115 120 125

tcc aac tcg ctc atc cac gcg ctc ctc gct gac ccc atc gat gcc gcg 432

Ser Asn Ser Leu Ile His Ala Leu Leu Ala Asp Pro Ile Asp Ala Ala

130 135 140

ccg cgt gac cct ttc gcg gac gcc gat gtc ggg tac cgg ccg gcg ctc 480

Pro Arg Asp Pro Phe Ala Asp Ala Asp Val Gly Tyr Arg Pro Ala Leu

145 150 155 160

cgg ttc ccg tgc tgc ctc ctc tgg gtc gac gtc gac gac gat gtt ctc 528

Arg Phe Pro Cys Cys Leu Leu Trp Val Asp Val Asp Asp Asp Val Leu

165 170 175

gac gaa tac ctc gac cgg cgc gtg gac gac atg gtc ggc gag ggg atg 576

Asp Glu Tyr Leu Asp Arg Arg Val Asp Asp Met Val Gly Glu Gly Met

180 185 190

gtc gag gag ctc gag gaa tac ttc gcg acg acg tcg gcc tcg gag cgg 624

Val Glu Glu Leu Glu Glu Tyr Phe Ala Thr Thr Ser Ala Ser Glu Arg

195 200 205

gcc tcg cac gcc ggg ctg ggc aag gcc atc ggc gtg ccg gag ctc ggc 672

Ala Ser His Ala Gly Leu Gly Lys Ala Ile Gly Val Pro Glu Leu Gly

210 215 220

gac tac ttc gcc ggg cgc aag agc ctc gac gcg gcg ata gac gag atc 720

Asp Tyr Phe Ala Gly Arg Lys Ser Leu Asp Ala Ala Ile Asp Glu Ile

225 230 235 240

aag gcc aac acg cgg gtc ctc gcg ggc cgc cag gtc ggc aag atc cga 768

Lys Ala Asn Thr Arg Val Leu Ala Gly Arg Gln Val Gly Lys Ile Arg

245 250 255

cgc at 773

Arg

<210>52

<211>257

<212>PRT

<213> Rice

<400>52

Met Glu Arg Ser Arg Val Gly Asp Gly Cys Cys Cys Ser Cys Ser Gly

1 5 10 15

Arg Gly Gly Val Ala Ser Thr Thr Ala Val Arg Pro Ser Thr Gly Met

20 25 30

Val Val Ile Val Gly Ala Thr Gly Thr Gly Lys Thr Lys Leu Ser Ile

35 40 45

Asp Ala Ala Gln Glu Leu Ala Gly Glu Val Val Asn Ala Asp Lys Ile

50 55 60

Gln Leu Tyr Asp Gly Leu Asp Val Thr Thr Asn Lys Val Ser Leu Ala

65 70 75 80

Asp Arg Arg Gly Val Pro His His Leu Leu Gly Ala Ile Arg Ala Glu

85 90 95

Ala Gly Glu Leu Pro Pro Ser Ser Ser Phe Arg Ser Leu Ala Ala Ala Ala

100 105 110

Ala Ala Gly Ile Ala Ser Arg Gly Arg Val Pro Val Val Ala Gly Gly

115 120 125

Ser Asn Ser Leu Ile His Ala Leu Leu Ala Asp Pro Ile Asp Ala Ala

130 135 140

Pro Arg Asp Pro Phe Ala Asp Ala Asp Val Gly Tyr Arg Pro Ala Leu

145 150 155 160

Arg Phe Pro Cys Cys Leu Leu Trp Val Asp Val Asp Asp Asp Val Leu

165 170 175

Asp Glu Tyr Leu Asp Arg Arg Val Asp Asp Met Val Gly Glu Gly Met

180 185 190

Val Glu Glu Leu Glu Glu Tyr Phe Ala Thr Thr Ser Ala Ser Glu Arg

195 200 205

Ala Ser His Ala Gly Leu Gly Lys Ala Ile Gly Val Pro Glu Leu Gly

210 215 220

Asp Tyr Phe Ala Gly Arg Lys Ser Leu Asp Ala Ala Ile Asp Glu Ile

225 230 235 240

Lys Ala Asn Thr Arg Val Leu Ala Gly Arg Gln Val Gly Lys Ile Arg

245 250 255

Arg

<210>53

<211>1284

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT7 genome sequence (34911308 aka NM_192112)

<221> CDS

<222>(1)...(1284)

<400>53

atg gca gcg act ggt cga aac gcg gcc gca cga cgg aca cgc cgc tcg 48

Met Ala Ala Thr Gly Arg Asn Ala Ala Ala Arg Arg Thr Arg Arg Ser

1 5 10 15

atc ccc cgc gcg gct gcc gtg ctc ccc ctc tcg tct gga tca ccg gcg 96

Ile Pro Arg Ala Ala Ala Val Leu Pro Leu Ser Ser Gly Ser Pro Ala

20 25 30

gct gtg ctg agg cga ctg ggg ctc ggt gag tgt ttt ggg tgg gcc ggc 144

Ala Val Leu Arg Arg Leu Gly Leu Gly Glu Cys Phe Gly Trp Ala Gly

35 40 45

ttt atg agc agt ctc ggt ttg aag atc cgc acc gtc gtc cgc tca cct 192

Phe Met Ser Ser Leu Gly Leu Lys Ile Arg Thr Val Val Arg Ser Pro

50 55 60

atg gcg gcc gcg gcc gtc gct ggc gtc gga agg gat ggt agc ttc gcc 240

Met Ala Ala Ala Ala Val Ala Gly Val Gly Arg Asp Gly Ser Phe Ala

65 70 75 80

tcc cag aag cgg cca cgt cgg gtt agt gtg aga atg gag aga agc aga 288

Ser Gln Lys Arg Pro Arg Arg Val Ser Val Arg Met Glu Arg Ser Arg

85 90 95

gtc ggg gac ggt tgc tgc tgc tcc tgc tct ggc cgc ggc ggg gtg gcg 336

Val Gly Asp Gly Cys Cys Cys Ser Cys Ser Gly Arg Gly Gly Val Ala

100 105 110

tcc act acg gcg gtc cgg ccg tcc acg ggg atg gtg gtg atc gtc ggc 384

Ser Thr Thr Ala Val Arg Pro Ser Thr Gly Met Val Val Ile Val Gly

115 120 125

gcc acg ggc acg ggg aag acc aag ctt tcc atc gac gcg gcg cag gag 432

Ala Thr Gly Thr Gly Lys Thr Lys Leu Ser Ile Asp Ala Ala Gln Glu

130 135 140

ctc gcc ggc gag gtg gtg aac gct gac aag att cag ctg tac gac ggc 480

Leu Ala Gly Glu Val Val Asn Ala Asp Lys Ile Gln Leu Tyr Asp Gly

145 150 155 160

ctc gac gtc acc acg aac aag gtg tcg ctc gcc gac cgc cgg ggc gtc 528

Leu Asp Val Thr Thr Asn Lys Val Ser Leu Ala Asp Arg Arg Gly Val

165 170 175

ccg cac cac ctc ctc ggc gca atc cgc gcc gag gcc ggg gag ctg ccg 576

Pro His His Leu Leu Gly Ala Ile Arg Ala Glu Ala Gly Glu Leu Pro

180 185 190

ccg tcg tcg ttc cgc tcg ctc gcc gcc gcc gcc gcg gcc ggc atc gcg 624

Pro Ser Ser Phe Arg Ser Leu Ala Ala Ala Ala Ala Ala Gly Ile Ala

195 200 205

tcg cgc ggg cgc gtg ccg gtc gtg gcc ggc ggg tcc aac tcg ctc atc 672

Ser Arg Gly Arg Val Pro Val Val Ala Gly Gly Ser Asn Ser Leu Ile

210 215 220

cac gcg ctc ctc gct gac ccc atc gat gcc gcg ccg cgt gac cct ttc 720

His Ala Leu Leu Ala Asp Pro Ile Asp Ala Ala Pro Arg Asp Pro Phe

225 230 235 240

gcg gac gcc gat gtc ggg tac cgg ccg gcg ctc cgg ttc ccg tgc tgc 768

Ala Asp Ala Asp Val Gly Tyr Arg Pro Ala Leu Arg Phe Pro Cys Cys

245 250 255

ctc ctc tgg gtc gac gtc gac gac gat gtt ctc gac gaa tac ctc gac 816

Leu Leu Trp Val Asp Val Asp Asp Asp Val Leu Asp Glu Tyr Leu Asp

260 265 270

cgg cgc gtg gac gac atg gtc ggc gag ggg atg gtc gag gag ctc gag 864

Arg Arg Val Asp Asp Met Val Gly Glu Gly Met Val Glu Glu Leu Glu

275 280 285

gaa tac ttc gcg acg acg tcg gcc tcg gag cgg gcc tcg cac gcc ggg 912

Glu Tyr Phe Ala Thr Thr Ser Ala Ser Glu Arg Ala Ser His Ala Gly

290 295 300

ctg ggc aag gcc atc ggc gtg ccg gag ctc ggc gac tac ttc gcc ggg 960

Leu Gly Lys Ala Ile Gly Val Pro Glu Leu Gly Asp Tyr Phe Ala Gly

305 310 315 320

cgc aag agc ctc gac gcg gcg ata gac gag atc aag gcc aac acg cgg 1008

Arg Lys Ser Leu Asp Ala Ala Ile Asp Glu Ile Lys Ala Asn Thr Arg

325 330 335

gtc ctc gcg gcc cgc cag gtc ggc aag atc cga cgc atg gcc gac gtt 1056

Val Leu Ala Ala Arg Gln Val Gly Lys Ile Arg Arg Met Ala Asp Val

340 345 350

tgg ggc tgg ccc atc cgc cgc ctc gac gcc acg gcc acc atc cgg gcg 1104

Trp Gly Trp Pro Ile Arg Arg Leu Asp Ala Thr Ala Thr Ile Arg Ala

355 360 365

cgg ctc tcc ggc gcc ggc cgc gcc gcc gag gcc gcc gcg tgg gag cgc 1152

Arg Leu Ser Gly Ala Gly Arg Ala Ala Glu Ala Ala Ala Trp Glu Arg

370 375 380

gac gtg cgc ggg cca ggc ctc gcc gcg atg cgt cag ttc gtc ggc cgc 1200

Asp Val Arg Gly Pro Gly Leu Ala Ala Met Arg Gln Phe Val Gly Arg

385 390 395 400

gcc gac ttc aac gcc gca gcg gtc gac cag cta gcc gcg cgg agt cgg 1248

Ala Asp Phe Asn Ala Ala Ala Val Asp Gln Leu Ala Ala Arg Ser Arg

405 410 415

agg caa tgc ctt cgc ggt ggc atg gtg gcc ggc tga 1284

Arg Gln Cys Leu Arg Gly Gly Met Val Ala Gly *

420 425

<210>54

<211>427

<212>PRT

<213> Rice

<400>54

Met Ala Ala Thr Gly Arg Asn Ala Ala Ala Arg Arg Thr Arg Arg Ser

1 5 10 15

Ile Pro Arg Ala Ala Ala Val Leu Pro Leu Ser Ser Gly Ser Pro Ala

20 25 30

Ala Val Leu Arg Arg Leu Gly Leu Gly Glu Cys Phe Gly Trp Ala Gly

35 40 45

Phe Met Ser Ser Leu Gly Leu Lys Ile Arg Thr Val Val Arg Ser Pro

50 55 60

Met Ala Ala Ala Ala Val Ala Gly Val Gly Arg Asp Gly Ser Phe Ala

65 70 75 80

Ser Gln Lys Arg Pro Arg Arg Val Ser Val Arg Met Glu Arg Ser Arg

85 90 95

Val Gly Asp Gly Cys Cys Cys Ser Cys Ser Gly Arg Gly Gly Val Ala

100 105 110

Ser Thr Thr Ala Val Arg Pro Ser Thr Gly Met Val Val Ile Val Gly

115 120 125

Ala Thr Gly Thr Gly Lys Thr Lys Leu Ser Ile Asp Ala Ala Gln Glu

130 135 140

Leu Ala Gly Glu Val Val Asn Ala Asp Lys Ile Gln Leu Tyr Asp Gly

145 150 155 160

Leu Asp Val Thr Thr Asn Lys Val Ser Leu Ala Asp Arg Arg Gly Val

165 170 175

Pro His His Leu Leu Gly Ala Ile Arg Ala Glu Ala Gly Glu Leu Pro

180 185 190

Pro Ser Ser Phe Arg Ser Leu Ala Ala Ala Ala Ala Ala Gly Ile Ala

195 200 205

Ser Arg Gly Arg Val Pro Val Val Ala Gly Gly Ser Asn Ser Leu Ile

210 215 220

His Ala Leu Leu Ala Asp Pro Ile Asp Ala Ala Pro Arg Asp Pro Phe

225 230 235 240

Ala Asp Ala Asp Val Gly Tyr Arg Pro Ala Leu Arg Phe Pro Cys Cys

245 250 255

Leu Leu Trp Val Asp Val Asp Asp Asp Val Leu Asp Glu Tyr Leu Asp

260 265 270

Arg Arg Val Asp Asp Met Val Gly Glu Gly Met Val Glu Glu Leu Glu

275 280 285

Glu Tyr Phe Ala Thr Thr Ser Ala Ser Glu Arg Ala Ser His Ala Gly

290 295 300

Leu Gly Lys Ala Ile Gly Val Pro Glu Leu Gly Asp Tyr Phe Ala Gly

305 310 315 320

Arg Lys Ser Leu Asp Ala Ala Ile Asp Glu Ile Lys Ala Asn Thr Arg

325 330 335

Val Leu Ala Ala Arg Gln Val Gly Lys Ile Arg Arg Met Ala Asp Val

340 345 350

Trp Gly Trp Pro Ile Arg Arg Leu Asp Ala Thr Ala Thr Ile Arg Ala

355 360 365

Arg Leu Ser Gly Ala Gly Arg Ala Ala Glu Ala Ala Ala Trp Glu Arg

370 375 380

Asp Val Arg Gly Pro Gly Leu Ala Ala Met Arg Gln Phe Val Gly Arg

385 390 395 400

Ala Asp Phe Asn Ala Ala Ala Val Asp Gln Leu Ala Ala Arg Ser Arg

405 410 415

Arg Gln Cys Leu Arg Gly Gly Met Val Ala Gly

420 425

<210>55

<211>5030

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT6 genome sequence (011783_3)

<221> CDS

<222>(1777)...(3031)

<400>55

acggagacga caagccacgg gaggacgacg tcggctatcg gacgagcttc ggggtgccct 60

aaggaaggag aagagaggga ttagagagag ggagatgaac cacggcgatc ggaaaccatg 120

gccgaaggcg gcggtggagg tggtgctcac ccgtgcgcga ggggatgggg gctccggcga 180

cgaacttcga cggaggaggg gtggacgagg tggatctcgg ccacgcgaat cgaacggcgg 240

cgacggcgca agacggcggc gagcctagcg gcggctagcg gcggccggag tagcggaaaa 300

aaacggcggc aaggggttgc acgacgcggg gacgatgggg agcacggggag aggtcggcgt 360

aatggggaaa aagagagagg gggtgccggc gatgcttaaa aagggggaga gagaaccgga 420

cgtggccggg aggggcggga atcgccggcc gacgtggggga gagggggagg agagaggc 480

gggattcgat tttcgaatcc cggccatctc gggcgcgggc gcgagcggga gagagaggga 540

agtgggcccg ggagacgcgg cgcacgcgcg ggcgtggtcg acgtggcccg gggaaggcgg 600

agacgtgggc gcggcgacgg ttgcgggcgc ggcggcgcgc tgggatcggc gggaggaggg 660

agacgggccc gacaggtggg ccccacctgt cggcgacccc gagagaggag acagcggggc 720

ggcctggctg ccggggctggg cctcggcctg cggccggccc agcagagagg agggggaagg 780

aaaggagaaa tgggccgacg gcccatttcg caaaaaggag gaaaaagaga ggaaaaaaaa 840

agaaaaagga aaaaggattt tccctggaat aaaatattgc ttgctcaatt ttaattggtt 900

aaaattattt ctagagctct gaaaattcca ctaaaaatcc tgttaatgaa tttcgacatg 960

tagaactcaa gaaaaattcc acatgtcaaa tccgattatt atttgcatta tttccttagg 1020

gttttctcct gatttcacct gcattttctt agggtcattt ataaaaatta caattttggc 1080

ttgggaggaa aacttcgggg tgtgacacgc ctctccccgt aactccgatg ggaggagccc 1140

cacgagccgc tccaccatgg catcgactcc aaccgccccc tctcctccct cccaggctca 1200

ccggccgcct ctccccccct ttccagccgc ccactgccgc tgccccctct ctcttaggct 1260

ccctatgccg ccggcagcct cggagaaaga gtagagaaga tactcgaata ggagaaagaa 1320

aagagaaaag aaaagcaaaa agcagtgtgg gtcccacatt ttttcttctc acttacatgt 1380

gggtcccata aattttttta ttttttgctg acacgtcagc aaaaccagag atcaatactg 1440

tatagggacc ttttttacac agttttgtat agtttagggc cgagatttct ggttttgtgg 1500

ttagagggcc ttaaaaaagc tcgctgttaa gttgagggac ctccagtgaa cttattccaa 1560

aatagaatgt ccaatttggg cctgaaagcc caatacttgt ttgtttgttt tgggcctcta 1620

catctgcaca ggctctcttt cagaaatccc atccatctcc tcctctatcc tcttcccttc 1680

ccttccaacac gaagccgccg cccgccggcc ggccgcccag aaccagaaag ctcctcctcc 1740

tcctcctccg cgcgccatca gatctcccag tgcggt atg cag tat gga tgc agg 1794

MET GLN TYR GLY CYS ARG

1 5

cgc ccc gcc gtg tgg aag aga agt tgg tcc ccg gct gcc gcc gcc gcc 1842

Arg Pro Ala Val Trp Lys Arg Ser Trp Ser Pro Ala Ala Ala Ala Ala

10 15 20

acc aag aac aag gtc atc gtc atc tca ggc ccc acc ggc gcc ggc aag 1890

Thr Lys Asn Lys Val Ile Val Ile Ser Gly Pro Thr Gly Ala Gly Lys

25 30 35

acc agg ctc gcc ttg gac ctc gcc aag agg ctc tcc ggg gag atc atc 1938

Thr Arg Leu Ala Leu Asp Leu Ala Lys Arg Leu Ser Gly Glu Ile Ile

40 45 50

agc gcc gac tcc gtc cag gtc tac cgg ggc ctc gac gtc ggc tcc gcc 1986

Ser Ala Asp Ser Val Gln Val Tyr Arg Gly Leu Asp Val Gly Ser Ala

55 60 65 70

aag ccc tcc tct tcc gac agg gcc gcc gtg ccg cac cac ctc atc gac 2034

Lys Pro Ser Ser Ser Asp Arg Ala Ala Val Pro His His Leu Ile Asp

75 80 85

atc ctc cac gcc tcc gac gac tac tcc gcc ggg gac ttc ttc cac gac 2082

Ile Leu His Ala Ser Asp Asp Tyr Ser Ala Gly Asp Phe Phe His Asp

90 95 100

gcc cgc gca gca acc gac cac ctc ctc gcc cga gcc cgc gtc ccc att 2130

Ala Arg Ala Ala Thr Asp His Leu Leu Ala Arg Ala Arg Val Pro Ile

105 110 115

gtc gcc gga ggg act ggc ctc tac ctc cgc tgg tac atc tat ggc aag 2178

Val Ala Gly Gly Thr Gly Leu Tyr Leu Arg Trp Tyr Ile Tyr Gly Lys

120 125 130

ccc agt gtc ccg cag tct tcc atg gac gtc acc tcc gcc gtc tgg tcc 2226

Pro Ser Val Pro Gln Ser Ser Met Asp Val Thr Ser Ala Val Trp Ser

135 140 145 150

gag ctc tcc cgc ttc cgg gac acc ggc cgc tgg gaa gaa gcc gtc gac 2274

Glu Leu Ser Arg Phe Arg Asp Thr Gly Arg Trp Glu Glu Ala Val Asp

155 160 165

ctg gtt gcc aac gcc ggc gac ccc aaa gct cgg gac ctg tca gtc aac 2322

Leu Val Ala Asn Ala Gly Asp Pro Lys Ala Arg Asp Leu Ser Val Asn

170 175 180

aac tgg tca agg tta agg aga agc ctt gag atc atc agg tct tca ggc 2370

Asn Trp Ser Arg Leu Arg Arg Ser Leu Glu Ile Ile Arg Ser Ser Gly

185 190 195

tca cct ccc tct gcc ttc tcc ttg ccc tac aat gct tac aat ctc aat 2418

Ser Pro Pro Ser Ala Phe Ser Leu Pro Tyr Asn Ala Tyr Asn Leu Asn

200 205 210

cac cac cgt cgt ctc agt ctc acc aac caa gcc gat caa ccc acg gag 2466

His His Arg Arg Leu Ser Leu Thr Asn Gln Ala Asp Gln Pro Thr Glu

215 220 225 230

ctg gag ctg gac tac gac ttc ctc tgc atc ttc ctc gcg tgc cca cgc 2514

Leu Glu Leu Asp Tyr Asp Phe Leu Cys Ile Phe Leu Ala Cys Pro Arg

235 240 245

gtt gag ctc tac aga tca atc gat ctg agg tgc gaa gag atg ctg gcc 2562

Val Glu Leu Tyr Arg Ser Ile Asp Leu Arg Cys Glu Glu Met Leu Ala

250 255 260

gac aca ggt ggc cta ctc tct gaa gcc tcc tgg ctc ctc gac atc ggc 2610

Asp Thr Gly Gly Leu Leu Ser Glu Ala Ser Trp Leu Leu Asp Ile Gly

265 270 275

ttg agt cct ggc atg aac tcg gct acc tgc gca atc ggc tsc agg caa 2658

Leu Ser Pro Gly Met Asn Ser Ala Thr Cys Ala Ile Gly Tyr Arg Gln

280 285 290

gcc atg gag tac ttg ctt cag tgt agg cac aac gga ggc agc agc tcc 2706

Ala Met Glu Tyr Leu Leu Gln Cys Arg His Asn Gly Gly Ser Ser Ser Ser

295 300 305 310

cca caa gag ttc ttg gag ttc ctg acc aag ttt cag act gcc tcc agg 2754

Pro Gln Glu Phe Leu Glu Phe Leu Thr Lys Phe Gln Thr Ala Ser Arg

315 320 325

aac ttc tca aag agg cag atg aca tgg ttc cgc aac gag aag att tac 2802

Asn Phe Ser Lys Arg Gln Met Thr Trp Phe Arg Asn Glu Lys Ile Tyr

330 335 340

cag tgg gtt gat gcc tcg cag cct ttc gac gcc att gcg cag ttt atc 2850

Gln Trp Val Asp Ala Ser Gln Pro Phe Asp Ala Ile Ala Gln Phe Ile

345 350 355

tgt gat gct tac cat gac cgt gct gca agg ctg gtt cct gat tca ctg 2898

Cys Asp Ala Tyr His Asp Arg Ala Ala Arg Leu Val Pro Asp Ser Leu

360 365 370

gaa atg aag agg gag agt tgc agg cac gag agc cgt gat ctc aag acc 2946

Glu Met Lys Arg Glu Ser Cys Arg His Glu Ser Arg Asp Leu Lys Thr

375 380 385 390

tac cgt tct gag aac agg gtg ttc cgt ggg gat gat gat tgt tgc cac 2994

Tyr Arg Ser Glu Asn Arg Val Phe Arg Gly Asp Asp Asp Cys Cys His

395 400 405

gtt ttg gat tgg atc aca agg aca cag agg aag tga c ctctactgct 3041

Val Leu Asp Trp Ile Thr Arg Thr Gln Arg Lys *

410 415

ctctcttgtt atctcttttc ccgccaggaa actatcctgc tgctgtgttt ggagatgtta 3101

caataggaaa tttcagcctt tttttgtcag aaaaatacag atacttgttt tgcaaatgtt 3161

ataccaaaag tgttttcaac tttggaattc aatgaaaatt cactagtgtt tcttcagcat 3221

atttctgtct gtattaactc tatgaaaaga agatatatga cattacagct tagagggtgt 3281

tttgcttgtg aatgtgagca tggagtttgc atggtctgaa actgaagttg gaagcatcac 3341

aaattttgtg agattcagag tactaaacag atgggagact tcacttcact tcacttgccg 3401

tgttacctgt atcactcttg ctcggttgtg gacgctgttt gtcaaccttc ttgtacttgt 3461

gctgcagcac ggcaatgtgg ttaatactaa tatttttgta tctgaaatct cccgtagtgc 3521

ttagtgctgt atttgtcaaa gcattgtacc cattactgaa gaagatgtgc aaaaccaaaa 3581

tgtgttgctg tctgcggtcg cattatcttc ctgctttccc ccaatcaaac actatctgca 3641

atttgcaatg gataagagat aaaaacaaaa catagcaaaa ggaagaaaaa gaaaacggcc 3701

gggagattga aagatgactt gttgggctac cagccatgtc tgatactctg atgcatcatg 3761

catatggtac tctcaagcaa ccaacgcaca agtggcagca gagtacatat aattctatcc 3821

ccttgtgtaa ggcaaccgtt ggcatctgat ctgagtatat tactacctcc gtattttaat 3881

gtatgacgcc gttgactttt cgacaacgtt tgaccattcg ttttattcta aaattttgtg 3941

taaatatgaa aatattatacg tcatgcttaa agaacatttg atgatgaatc aagtcataat 4001

aaaataaata ataattacat aaatttttcg aataagacga atggtcaaac attgggaaaa 4061

aaagtcaacg tcatcataca ttaaaatatg gaggtagtac aacatattgt gccagaagaa 4121

gttcctctaa ttacccaaac tgacatggca tatgttgccc tgaaacatgt tgtaaggcaa 4181

ggtcaatgga ttcaattgga agtcagatta aggattaagt agttagcaca actgcaattg 4241

aaggaccaga atatatacaa ggttgaatga ttgcacgtgc tctggactag ctttctggtc 4301

agctctttgg acggtcacaa gctctgaata taacaactgg tgcactttct tgctgatcga 4361

tcattagtca gggatccaac gttgccaatg atctgtagca caactaagga gatcatcatc 4421

atatatatga tggtgcggtt gtaaccgcgt ctgcacctaa ctaaggaaaa agaaacatgt 4481

cgttttcggc tgtagcagag aaagttgcag gacaacccaa aatatgtata gccaacagaa 4541

atactgttcc aaatcttatg gttatatgca atgataccca ccaaacattt tggagggatg 4601

cactcgacat aatttgctct ccatgaaaaa ggcaggagag aaggaaaatg cagaaaagac 4661

agtgcaaaag cagtggtgtc atgtgtgtgc ccaccttgat gtaaagcacc agctcttata 4721

tagcttcaaa agcgtgaaga ttctttgtca aacagcaagg taagtactag tgcttgtcaa 4781

tactttttga agaaaatagg cactccaagg agcagcattt gttttatgcc tacatgctta 4841

ctcttgctat acgagtacat atgagtagta catcatttca ggcctgcttg ctagagctgc 4901

aagaaggaag gagccgcaga tgctcacaag tcatgattaa ggattaatat taatgccgaa 4961

gcttttgcta ggtcttcatc ttttcctttt aaattttttt tggggggggga gatagagggg 5021

attgatctg 5030

<210>56

<211>1254

<212>DNA

<213> Rice

<220>

<221> CDS

<222>(1)...(1254)

<221>misc_feature

<222>(0)...(0)

<223> OsIPT6 coding sequence

<400>56

atg cag tat gga tgc agg cgc ccc gcc gtg tgg aag aga agt tgg tcc 48

Met Gln Tyr Gly Cys Arg Arg Pro Ala Val Trp Lys Arg Ser Trp Ser

1 5 10 15

ccg gct gcc gcc gcc gcc acc aag aac aag gtc atc gtc atc tca ggc 96

Pro Ala Ala Ala Ala Ala Thr Lys Asn Lys Val Ile Val Ile Ser Gly

20 25 30

ccc acc ggc gcc ggc aag acc agg ctc gcc ttg gac ctc gcc aag agg 144

Pro Thr Gly Ala Gly Lys Thr Arg Leu Ala Leu Asp Leu Ala Lys Arg

35 40 45

ctc tcc ggg gag atc atc agc gcc gac tcc gtc cag gtc tac cgg ggc 192

Leu Ser Gly Glu Ile Ile Ser Ala Asp Ser Val Gln Val Tyr Arg Gly

50 55 60

ctc gac gtc ggc tcc gcc aag ccc tcc tct tcc gac agg gcc gcc gtg 240

Leu Asp Val Gly Ser Ala Lys Pro Ser Ser Ser Asp Arg Ala Ala Val

65 70 75 80

ccg cac cac ctc atc gac atc ctc cac gcc tcc gac gac tac tcc gcc 288

Pro His His Leu Ile Asp Ile Leu His Ala Ser Asp Asp Tyr Ser Ala

85 90 95

ggg gac ttc ttc cac gac gcc cgc gca gca acc gac cac ctc ctc gcc 336

Gly Asp Phe Phe His Asp Ala Arg Ala Ala Thr Asp His Leu Leu Ala

100 105 110

cga gcc cgc gtc ccc att gtc gcc gga ggg act ggc ctc tac ctc cgc 384

Arg Ala Arg Val Pro Ile Val Ala Gly Gly Thr Gly Leu Tyr Leu Arg

115 120 125

tgg tac atc tat ggc aag ccc agt gtc ccg cag tct tcc atg gac gtc 432

Trp Tyr Ile Tyr Gly Lys Pro Ser Val Pro Gln Ser Ser Met Asp Val

130 135 140

acc tcc gcc gtc tgg tcc gag ctc tcc cgc ttc cgg gac acc ggc cgc 480

Thr Ser Ala Val Trp Ser Glu Leu Ser Arg Phe Arg Asp Thr Gly Arg

145 150 155 160

tgg gaa gaa gcc gtc gac ctg gtt gcc aac gcc ggc gac ccc aaa gct 528

Trp Glu Glu Ala Val Asp Leu Val Ala Asn Ala Gly Asp Pro Lys Ala

165 170 175

cgg gac ctg tca gtc aac aac tgg tca agg tta agg aga agc ctt gag 576

Arg Asp Leu Ser Val Asn Asn Trp Ser Arg Leu Arg Arg Ser Leu Glu

180 185 190

atc atc agg tct tca ggc tca cct ccc tct gcc ttc tcc ttg ccc tac 624

Ile Ile Arg Ser Ser Gly Ser Pro Pro Ser Ala Phe Ser Leu Pro Tyr

195 200 205

aat gct tac aat ctc aat cac cac cgt cgt ctc agt ctc acc aac caa 672

Asn Ala Tyr Asn Leu Asn His His Arg Arg Leu Ser Leu Thr Asn Gln

210 215 220

gcc gat caa ccc acg gag ctg gag ctg gac tac gac ttc ctc tgc atc 720

Ala Asp Gln Pro Thr Glu Leu Glu Leu Asp Tyr Asp Phe Leu Cys Ile

225 230 235 240

ttc ctc gcg tgc cca cgc gtt gag ctc tac aga tca atc gat ctg agg 768

Phe Leu Ala Cys Pro Arg Val Glu Leu Tyr Arg Ser Ile Asp Leu Arg

245 250 255

tgc gaa gag atg ctg gcc gac aca ggt ggc cta ctc tct gaa gcc tcc 816

Cys Glu Glu Met Leu Ala Asp Thr Gly Gly Leu Leu Ser Glu Ala Ser

260 265 270

tgg ctc ctc gac atc ggc ttg agt cct ggc atg aac tcg gct acc tgc 864

Trp Leu Leu Asp Ile Gly Leu Ser Pro Gly Met Asn Ser Ala Thr Cys

275 280 285

gca atc ggc tac agg caa gcc atg gag tac ttg ctt cag tgt agg cac 912

Ala Ile Gly Tyr Arg Gln Ala Met Glu Tyr Leu Leu Gln Cys Arg His

290 295 300

aac gga ggc agc agc tcc cca caa gag ttc ttg gag ttc ctg acc aag 960

Asn Gly Gly Ser Ser Ser Ser Pro Gln Glu Phe Leu Glu Phe Leu Thr Lys

305 310 315 320

ttt cag act gcc tcc agg aac ttc tca aag agg cag atg aca tgg ttc 1008

Phe Gln Thr Ala Ser Arg Asn Phe Ser Lys Arg Gln Met Thr Trp Phe

325 330 335

cgc aac gag aag att tac cag tgg gtt gat gcc tcg cag cct ttc gac 1056

Arg Asn Glu Lys Ile Tyr Gln Trp Val Asp Ala Ser Gln Pro Phe Asp

340 345 350

gcc att gcg cag ttt atc tgt gat gct tac cat gac cgt gct gca agg 1104

Ala Ile Ala Gln Phe Ile Cys Asp Ala Tyr His Asp Arg Ala Ala Arg

355 360 365

ctg gtt cct gat tca ctg gaa atg aag agg gag agt tgc agg cac gag 1152

Leu Val Pro Asp Ser Leu Glu Met Lys Arg Glu Ser Cys Arg His Glu

370 375 380

agc cgt gat ctc aag acc tac cgt tct gag aac agg gtg ttc cgt ggg 1200

Ser Arg Asp Leu Lys Thr Tyr Arg Ser Glu Asn Arg Val Phe Arg Gly

385 390 395 400

gat gat gat tgt tgc cac gtt ttg gat tgg atc aca agg aca cag agg 1248

Asp Asp Asp Cys Cys His Val Leu Asp Trp Ile Thr Arg Thr Gln Arg

405 410 415

aag tga 1254

Lys *

<210>57

<211>417

<212>PRT

<213> Rice

<400>57

Met Gln Tyr Gly Cys Arg Arg Pro Ala Val Trp Lys Arg Ser Trp Ser

1 5 10 15

Pro Ala Ala Ala Ala Ala Thr Lys Asn Lys Val Ile Val Ile Ser Gly

20 25 30

Pro Thr Gly Ala Gly Lys Thr Arg Leu Ala Leu Asp Leu Ala Lys Arg

35 40 45

Leu Ser Gly Glu Ile Ile Ser Ala Asp Ser Val Gln Val Tyr Arg Gly

50 55 60

Leu Asp Val Gly Ser Ala Lys Pro Ser Ser Ser Asp Arg Ala Ala Val

65 70 75 80

Pro His His Leu Ile Asp Ile Leu His Ala Ser Asp Asp Tyr Ser Ala

85 90 95

Gly Asp Phe Phe His Asp Ala Arg Ala Ala Thr Asp His Leu Leu Ala

100 105 110

Arg Ala Arg Val Pro Ile Val Ala Gly Gly Thr Gly Leu Tyr Leu Arg

115 120 125

Trp Tyr Ile Tyr Gly Lys Pro Ser Val Pro Gln Ser Ser Met Asp Val

130 135 140

Thr Ser Ala Val Trp Ser Glu Leu Ser Arg Phe Arg Asp Thr Gly Arg

145 150 155 160

Trp Glu Glu Ala Val Asp Leu Val Ala Asn Ala Gly Asp Pro Lys Ala

165 170 175

Arg Asp Leu Ser Val Asn Asn Trp Ser Arg Leu Arg Arg Ser Leu Glu

180 185 190

Ile Ile Arg Ser Ser Gly Ser Pro Pro Ser Ala Phe Ser Leu Pro Tyr

195 200 205

Asn Ala Tyr Asn Leu Asn His His Arg Arg Leu Ser Leu Thr Asn Gln

210 215 220

Ala Asp Gln Pro Thr Glu Leu Glu Leu Asp Tyr Asp Phe Leu Cys Ile

225 230 235 240

Phe Leu Ala Cys Pro Arg Val Glu Leu Tyr Arg Ser Ile Asp Leu Arg

245 250 255

Cys Glu Glu Met Leu Ala Asp Thr Gly Gly Leu Leu Ser Glu Ala Ser

260 265 270

Trp Leu Leu Asp Ile Gly Leu Ser Pro Gly Met Asn Ser Ala Thr Cys

275 280 285

Ala Ile Gly Tyr Arg Gln Ala Met Glu Tyr Leu Leu Gln Cys Arg His

290 295 300

Asn Gly Gly Ser Ser Ser Ser Pro Gln Glu Phe Leu Glu Phe Leu Thr Lys

305 310 315 320

Phe Gln Thr Ala Ser Arg Asn Phe Ser Lys Arg Gln Met Thr Trp Phe

325 330 335

Arg Asn Glu Lys Ile Tyr Gln Trp Val Asp Ala Ser Gln Pro Phe Asp

340 345 350

Ala Ile Ala Gln Phe Ile Cys Asp Ala Tyr His Asp Arg Ala Ala Arg

355 360 365

Leu Val Pro Asp Ser Leu Glu Met Lys Arg Glu Ser Cys Arg His Glu

370 375 380

Ser Arg Asp Leu Lys Thr Tyr Arg Ser Glu Asn Arg Val Phe Arg Gly

385 390 395 400

Asp Asp Asp Cys Cys His Val Leu Asp Trp Ile Thr Arg Thr Gln Arg

405 410 415

Lys

<210>58

<211>8306

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT10 genome sequence (011410_2)

<221> exon

<222>(2001)...(2675)

<221> exons

<222>(3415)...(3438)

<221> exon

<222>(3922)...(4065)

<221> exon

<222>(5392)...(6306)

<400>58

tagagacata tgggaagatc tctcaccgag aaactgatcc atgagtttaa gcaaaactgg 60

tcattataat aaagaaaaat ctacaaaatt actgttgtta attcccttga actactgtca 120

tcaagaaaga ttggttgatg atggacatgt ccgcagcaat ggagggtcac cgcgtcgtct 180

ccatcggcag cgagatctgc gagcaggatc acgccggcgg cgaccagcgc tccaccgccg 240

gcgacgatgg cgacggcgac ggtgacggcc ccttacgtctc cctcttcgag ctcgcgccca 300

tcgtggcgcg cgcaccgcag gacgaggacg gacacggcca ggaagacagc catgcccaag 360

aggtgttcga tgacctgccg gccgagctgc ggcgcgacgg cgacggcgcg ctgaccgtgg 420

gcgggctcgc agcggcgctc cgggcgcagc ggagggagct ggaggccgtt cgcgccgagc 480

tcgacggcga gcggcgcgcg ggcgcggagg cggcggagta ccagcggcag ctggaggagc 540

aggggggagtt cgaccggggag gcggtgcgcc tcgccatgca gctcgtccac gaggccgaga 600

cggagaagca cgccctgcag caccagctcg acgcgttccg ggtcaaggcc cagctctacg 660

actacgaggc cgccgccacc gccgccgcca gggaccacga cgcggccggc gacggcggcg 720

gcggcggcaa caactaccag tcgctggtgg acttcttgcc ggggtcggtg ttctcctcct 780

cgccggacct ggccaacctg ctcaagctct acaccgaggg caatggcggc ggccgtcgac 840

tgaccgacgc gccggtgccg gtggtcaccg aggtggttga ggaagaagag gaagaggagg 900

aggagggagga ggaggaagtc gccgtcgcgg ccatcggtgg cgtggattcg aacgggaacg 960

gtggtgctgc cgccaccatc gccatcgccg gcgattcttt gcaggaagga agcagtgatc 1020

acctcgaacc cacagaagtg tcaccgcaac ggtgaattgt gaatcttggg attaatacaa 1080

ccttgtcaat atatacatat catgtaatta tatttgacta gccacatgtg cccgcgcttc 1140

gttgcggatc atgaatttgt aaattaccgt agtaaagaaa gttttttcta atatgtatac 1200

tgatccattg tttaaatggc tgcaatatat tttaattactt aatgataccc cacgcgttgt 1260

tgcagaaaat tctcaaattt tagttatgg gtagaatgta caacctaatg ctaaaaagtt 1320

agagaaacaa aatgattttt tggaccaata attgtgtgaa tagcgtaagt caaattctaa 1380

aaataattta ggtaacacat tttagcagaa acttcaaaag atgtacatac tttgaggtca 1440

tgagttaaca tacaatgttg tactgtatta ttcatcaaat attgtcacaa tgtagcttac 1500

tcatagatat aagattgtat gtatctagtg agggacaatt tatattgttc actaacatct 1560

aatattgttc tcggaatggt atccagagaa atttttatga tttagaataa aatggaacaa 1620

tgttgtataa atctataaaa atataattgc ttaattattt atatgcttag atttggcacc 1680

tctaaatgtg gcctaactac cattggacca cagtaagtag gggctcataa agatgcatcc 1740

cctataaaag ccaagggaca ccgagagtcc tctacggaag aattctaccc ttcccattag 1800

gacagtcaaa caccttattg ctactccaat cttcctttca gtatggagaa ctcctcaaag 1860

aaaacccaag agttcttccc taaaggtggg aatggaggtt atgctgagca gctggatctc 1920

ttgctgaagc agcttcgttt tcctaacaac cgatccacca tgcggagcaa gtgatcaaag 1980

gattccagaa ggattggacg atgaagatct aattcaagc cagggaagag aagtgtcaag 2040

gacatgtgtt caagtcccgc caccttcgag ccaacaaaga ggcagcactc caggatgcgt 2100

cgcgtgaggc attcatgcgt ctatgtaaga tctacagcat cgaggttgcg agtactccgt 2160

tctttctaca tccattccgt gaatgcggtg accgccgctg ccatattcgg aaatttaggg 2220

gctttgagga gcactctccc atccacttct ccatgtggat gtgggctgca gacgaggcct 2280

atgaggaggc cttagaggaa ttagatatgc ttcggtcaaa gattgccggc tgggaggagc 2340

ggtacaacca ccttgctaaa gaacacacca ctcgtggaca actattggaa gcaatcaagc 2400

ttcgcctcca gtggtatttt cgaaccccat ctcaagctca aatccaacgg actttgtcac 2460

caccaccaca aagagtgaca agaagtgatg gtgaggacta tagtcaaatc aatgcacagc 2520

aggcatgtct ggaaaggtcc gaagttaaac ttgatagggc aacttcacaa gactatctgc 2580

aaggatacaa gcccccatca gaatccctcg acgctattgt ttggcctctt gttgaaggga 2640

agcatgacaa tacaagcagc ggtagagga atgaggtaaa ggaaactgct cacaataacc 2700

aagggaccct gttgggctag tcctcggaaa gagagttgga ccagctacat atctagaaga 2760

ccacaatgta agtgacaggg ctatatcttg tcacgtagga gtagcatgtg ggtgggagtt 2820

ggaccaattt cacatatagg agaccgctat gtaagtgaca ggttatagcc tgtcacctag 2880

cagtactatg tgggtggtca agatcaccta tcaagtgtgc ttgtctatgc ctagttgtga 2940

cctaccagtt agagtagtat gtgagggtgg tagtaagatt gtattccctt tgtccagttg 3000

tgggtggaca agctaggcgg atagtctagt gtgtttgtgt atgcgtggtt gtgatgcttt 3060

tgtgcttggc ccgaggaca tgagcaatat ttgcttaaaa gtgcttgttt tcttctgcaa 3120

tgctactttg ttttcatgat catgcaagtt acctaaatac atgtgaattg ttctagttga 3180

tgggatctat tgcgatagaa tcaaatgatt tccaattgta tagtaacgga gctagcaaca 3240

gtaatataac cattttgacc aggatggttc aaaagtaaac catatagaaa aggagttgtt 3300

tattaaatat atgtattgta tcaactaaaa tagtacacaa tggccaataa ttttgcaatg 3360

aatttagttg ataattggca tggtatggtt tttttttttt tttgcttttt gcagaaggca 3420

tgggaaatgg caaaacaagt aaatatatta caaagtaatt tctaacgatt gttagtaacc 3480

ggaagatggt tggtattaga ttaccaagtt tggaagtatt attttaccag agaacgtata 3540

agtaacatgt atattgttcg aagtgcccac atttgaattt attcgatg aaggattgtt 3600

atgtaatttt tccttgaaaa atgtgcaaaa gcacatgttt acaaatcatc accatatctt 3660

aagatgaaag taggcataag gtttaaaaag tcaaaggtaa ttattagggtt tatttttttg 3720

ttatgcttac acacgtattg acgatacaaa ggtttgagcc attaactctg atgcctaaaa 3780

atatcttcaa agaaaaaaaa tcgatcacaa ttgcttgaat aatagtaaga gtatacctaa 3840

ctgaatttgg ggcccaccaa tataattacc gtacacatcc aactgcacat ggattgatag 3900

gccaaattac tataattgga ggtgcctaga ggaaggattt atgctttggt ctataaacat 3960

caagacaata ttggactggt ccttgaaaag agagttggac cagctgcaca tctaggagac 4020

cgctatgtaa gtgacagggt catatcacat ctaggagacc tctatgtaag tgacagggtc 4080

atatgttgtc acctaggagt agtatgtggg taagagttgg accaacttca catataggta 4140

accgctatgt aagtgatagg gtcatagcct gtcacctagc agcactatgt gggtgatcaa 4200

aatggcctct ctggtgtgct tgtctatgcc taattgtaat gcttttgtgc ttggctcgag 4260

gccaatgagt aatgtatgct tgaactgcta tgtaagcgac agggtcatag cttattagtt 4320

agagtagtag gtgagggtgg taataaaatt gtattccctt tgtctagtta tgggtggaca 4380

aggtgggtaa tctagtgtgt ttgtgtatgc gtggttgtga tgcttttgtg cttggcccga 4440

ggataatgag caatatttgc ttaaagttgc atgttttctt ctccaatgca ggtttgtttt 4500

catgagcatg caaattatct aaatacatgt gaattgttct agttgatggg atctattgcg 4560

atagaatcaa atggcctcta atcgtatagt aacagagcta gcaacagtaa tataaccatc 4620

ttaaccagga tggttcaaaa gcaaaccata tataaaagga gttgtttatt aaatatatgt 4680

attgtatcaa ccggaatatt acacaagggc taataatttt gcaatgaatt tatttgataa 4740

ttggcattgt atggctattt tgttttttgc attttgcgga aggcatgaga aatgccaaaa 4800

caagtaaata tagagcaaag tattttacaa cgattgttag taagtatttt tgaggtagat 4860

ggttggtatt atattaccaa gtttcaaagt attcttttac cagagaacat ataagtaaca 4920

tatgtatgat ttgaattgcc cacatttgaa tttactttcg atgaaggatt gatacggatt 4980

ttttttcctt gaaaaatgtg taaaagcaca tgtttacaaa tcatcaccat attaagatga 5040

aagtaggcat attgtttaaa aagttaaagg agcttatcag gtttaatttg ttttatgctt 5100

acacacgtat tgaggataca attttaaggg ttgagccgtt aactcttatg ccaaaaatat 5160

ctccaaagaa aaaaaatcga tcacaattgc ttggataatt gtatgagtat atctaattga 5220

atttgggccc catcaagatg attaccatac attcaact gtacatggat tgatatgcca 5280

aattccggta agtggaggtg ccaagaggaa ggaggaagga tttatgcttt gatctagaaa 5340

catcaaggcg gcacactttc ccctttccta tatactgagg aactcttcca ggtaatacga 5400

acccttagct actttccttt catgctcaat tttcaccctt cttgtgattg cttcctcaat 5460

atgctgggaa acaagttagt agtgattatt ggtgccacgg gaactggaaa aacaagactc 5520

tcaattgaga ttgccaaggc gattggtggg gaagtggtaa atggtgacaa gatgcaaatt 5580

tatgatggcc tggatattac gacaaacaag gtttctttac aagatcgatg cggcatacct 5640

catcacctta ttgcgtccat ccctcacaac gcaggtgatt ttcctgtgtc attttttcga 5700

tatgctgcaa aaaccacaat aaactgcatt gccagacgtg gtcacacacc gattgtggtg 5760

ggtggatcta actcacttat ccatggtctc cttgttgaca attttgattt gtctattgtg 5820

gatccttttg ggcaattgga ggttagctat cagccgacgc ctcaatggca atgttgtttt 5880

ctatgggttc atgttaatga ggtgattctt aatgagtatt tgaaacgtcg tgttgacggc 5940

atggttgatg ctgggttagt tgaggaaatt gaagaatatt ttgacacatt atcagttaat 6000

ggacatgttc catatgtggg attagggaag gccattggtg ttccagagct aagcgagtat 6060

tttactggac gggtgagttg tagtgatgct ctttctatga tgaagaccaa tacacagatt 6120

cttgcacgat ctcaagtcac aaagattcat cgcatggttg atgtgtgggg atggcatgtt 6180

catgcccttg attgtactga aactattcta gcacatctta ctggatcaaa taagtatatg 6240

gaggatttgg tgtggaaacg tgatgtaagt gactctggac ttgctgctat acaagatttt 6300

ctgtgataat atcagaagat gggaagctag tttctcaaac acatcggcta ttgattttgt 6360

ctacaataat ggtttaatcg tctggcttgc ttagtaattt tacagatcat ggcatactaa 6420

gttaacttgg atcattttgg gtgtgtttgg aaggagcaaa cgtcaattgg tgtatatgaa 6480

attacktgga ggccttttgt accttaaaca cttggatgcc ttttatttta cataatagtt 6540

atatatagtt gttgttcata attttttgat gtcatcaata ttcatacgtg gtgatgcaat 6600

tcttattgat tatctctaat agatatgatg tcgtgccaac aaaaacaaca aacatggaag 6660

tcacaaatag tcatataaga aaataataga gggttcccag ttgttcatgc accaagctta 6720

atacaaatag gaaataaaca tgatagtcca atgacaatgg accaagttta gagtagcacc 6780

acacacaatg cttgttcact tactgataca acataaataa taaagagtta agtatgacaa 6840

cacaaaaaac atcccctgca acaaagagcc cacatagaga gtatacataa aatccaaaaa 6900

caatgttttt gttaaatctc tggttgggaa gtaattattt gtcattacag tcgaaatttt 6960

caaacttgaa aacttaacca taggaatttt tggagagccc agcctttgag gatggactta 7020

gaatttggag gaaattttct aagaggtga taaacccaaa cctcaagatt caaatatttg 7080

gatcaagact tttgggcttg ggatttggtg tttgaagaaa cagcgggatt tgagagtact 7140

ggcacataat cctaaataca ctcaaagaat caaaagattt taaacatagg tttcaaataa 7200

aaaaaatcaa ccgaggcaaa acccaaggcg ttgcaatcct accccctatt aatagaatct 7260

cgtcttgaga tttcggccaa agaagggtag cagaatgttg ttgtggctcc tgttcagtga 7320

taggctcaat accagggaca tgttggatag aagacattgt gcaaaggaag atgatgatct 7380

aacatgtgtt gtgtgtaatg gtgagtgtag agaaactcgg cttcatctct tttctgccta 7440

ccctagcatc agatgtaggc aacacttggg aattgaatgg aaacataacc tggaattttt 7500

cccaacggtt gttctcgcga gattgaggtt tggtcggaga ggttttctgg aaatattttt 7560

tatagcctca tggcatattt ggaaacagag aaagaggctt attttccaaa atatcctgcc 7620

tatgttccag tcttggaggt tgctttttgt gaatgaagtt cttctacata tgtgtagaat 7680

gaaggatcct ctaaaacaat ctgtttttga ttggttacaa acctatagg ttttgagttt 7740

tccctgtaat tgttttaatg aaaatacact gctaggcaaa gccctggcag tatttgcagt 7800

taaaaaaata gggtccttga aatgatacat ggtctatgtg ctgacctttt cctttggtga 7860

ttgaggcatt cctatcccat cttttactga gtgatacatg ggccactgtt tgacccaaaa 7920

tttttgaact caagtgtcga ctctgaactg atactgtctt tgttgagaat ctaaagttct 7980

tctttggtgt cagtgctggt gttgttatgt cctgatcggg caataatggg gacctctatt 8040

tggacgtgtt gtggccatat cctgcatcct tgccggttgt tatgatagtc attcggggta 8100

ttcgagatca tttctcagcc tctatacatg ttcaccaaca tacttttttt ttacctcgtg 8160

cacttgggta cccatttcat tgagcgcacc cttatcttcg ggggcccatc tctgtctcct 8220

tggagtggtt ttggtcgtgc acgcgggtgt ggcgagtgga ggcggtgtag ctcgggcgag 8280

gcgaaaaaat ggagcggagg ttcgac 8306

<210>59

<211>585

<212>PRT

<213> Rice

<220>

<221>CHAIN

<222>(0)...(0)

<223> OsIPT10 amino acid sequence (011410_2)

<400>59

Met Lys Ile Tyr Ile Gln Ala Arg Glu Glu Lys Cys Gln Gly His Val

1 5 10 15

Phe Lys Ser Arg His Leu Arg Ala Asn Lys Glu Ala Ala Leu Gln Asp

20 25 30

Ala Ser Arg Glu Ala Phe Met Arg Leu Cys Lys Ile Tyr Ser Ile Glu

35 40 45

Val Ala Ser Thr Pro Phe Phe Leu His Pro Phe Arg Glu Cys Gly Asp

50 55 60

Arg Arg Cys His Ile Arg Lys Phe Arg Gly Phe Glu Glu His Ser Pro

65 70 75 80

Ile His Phe Ser Met Trp Met Trp Ala Ala Asp Glu Ala Tyr Glu Glu

85 90 95

Ala Leu Glu Glu Leu Asp Met Leu Arg Ser Lys Ile Ala Gly Trp Glu

100 105 110

Glu Arg Tyr Asn His Leu Ala Lys Glu His Thr Thr Arg Gly Gln Leu

115 120 125

Leu Glu Ala Ile Lys Leu Arg Leu Gln Trp Tyr Phe Arg Thr Pro Ser

130 135 140

Gln Ala Gln Ile Gln Arg Thr Leu Ser Pro Pro Pro Gln Arg Val Thr

145 150 155 160

Arg Ser Asp Gly Glu Asp Tyr Ser Gln Ile Asn Ala Gln Gln Ala Cys

165 170 175

Leu Glu Arg Ser Glu Val Lys Leu Asp Arg Ala Thr Ser Gln Asp Tyr

180 185 190

Leu Gln Gly Tyr Lys Pro Pro Ser Glu Ser Leu Asp Ala Ile Val Trp

195 200 205

Pro Leu Val Glu Gly Lys His Asp Asn Thr Ser Ser Gly Arg Arg Asn

210 215 220

Glu Lys Ala Trp Glu Met Ala Lys Gln Val Pro Arg Gly Arg Ile Tyr

225 230 235 240

Ala Leu Val Tyr Lys His Gln Asp Asn Ile Gly Leu Val Leu Glu Lys

245 250 255

Arg Val Gly Pro Ala Ala His Leu Gly Asp Arg Tyr Val Ser Asp Arg

260 265 270

Val Ile Ser His Leu Gly Asp Leu Tyr Val Ile Arg Thr Leu Ser Tyr

275 280 285

Phe Pro Phe Met Leu Asn Phe His Pro Ser Cys Asp Cys Phe Leu Asn

290 295 300

Met Leu Gly Asn Lys Leu Val Val Ile Ile Gly Ala Thr Gly Thr Gly

305 310 315 320

Lys Thr Arg Leu Ser Ile Glu Ile Ala Lys Ala Ile Gly Gly Glu Val

325 330 335

Val Asn Gly Asp Lys Met Gln Ile Tyr Asp Gly Leu Asp Ile Thr Thr

340 345 350

Asn Lys Val Ser Leu Gln Asp Arg Cys Gly Ile Pro His His Leu Ile

355 360 365

Ala Ser Ile Pro His Asn Ala Gly Asp Phe Pro Val Ser Phe Phe Arg

370 375 380

Tyr Ala Ala Lys Thr Thr Ile Asn Cys Ile Ala Arg Arg Gly His Thr

385 390 395 400

Pro Ile Val Val Gly Gly Ser Asn Ser Leu Ile His Gly Leu Leu Val

405 410 415

Asp Asn Phe Asp Leu Ser Ile Val Asp Pro Phe Gly Gln Leu Glu Val

420 425 430

Ser Tyr Gln Pro Thr Pro Gln Trp Gln Cys Cys Phe Leu Trp Val His

435 440 445

Val Asn Glu Val Ile Leu Asn Glu Tyr Leu Lys Arg Arg Val Asp Gly

450 455 460

Met Val Asp Ala Gly Leu Val Glu Glu Ile Glu Glu Tyr Phe Asp Thr

465 470 475 480

Leu Ser Val Asn Gly His Val Pro Tyr Val Gly Leu Gly Lys Ala Ile

485 490 495

Gly Val Pro Glu Leu Ser Glu Tyr Phe Thr Gly Arg Val Ser Cys Ser

500 505 510

Asp Ala Leu Ser Met Met Lys Thr Asn Thr Gln Ile Leu Ala Arg Ser

515 520 525

Gln Val Thr Lys Ile His Arg Met Val Asp Val Trp Gly Trp His Val

530 535 540

His Ala Leu Asp Cys Thr Glu Thr Ile Leu Ala His Leu Thr Gly Ser

545 550 555 560

Asn Lys Tyr Met Glu Asp Leu Val Trp Lys Arg Asp Val Ser Asp Ser

565 570 575

Gly Leu Ala Ala Ile Gln Asp Phe Leu

580 585

<210>60

<211>7608

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223>OsIPT9 genome sequence (005021_3)

<221> exon

<222>(2001)...(3079)

<221> exon

<222>(3322)...(3390)

<221> exon

<222>(4363)...(4436)

<221> exon

<222>(5516)...(5608)

<400>60

taatgttatg atctgctcgc tccattaaa agtcttaagc atatttttta tattttttaa 60

taaatgagat ggtcatattt atattcaaga cttaaatgta gagattatct tttcatattg 120

tcgcagtgct aaattaaatt attgatccga acccaagtag ttatcttaat atatatttat 180

atgttgtata tattagtgta gcttttttta atttgttaca tgtgcattga gttgattttt 240

tcatggtttt gattatagaa aatgagaaaa ttgtaatatt gtgacctaaa ttttttggat 300

agaggggagta tgaccggtga aatctccaaa actactgcag ggtagagta ctcgtagtac 360

ttctaaccag cctgaccaca ttcaaacgca cagtagtaca gtacttggag gacattcaag 420

tcacagtaca caaatgtttc cagcacaata aaactacttg gaacagtact gtgggcagca 480

gctctttgca ggccgcaggc gagtacagat ggaccctcat cgaaacgaaa cgaaacccat 540

gccaagaacc tgcgtgctct ttgatgagcc agtcattcct cgaggcatct tttcatcaga 600

tcagaattca gagcctcgcg agaaagaaag gctggcccaa tcctgcagct gcacagatcc 660

cagcagctgc agctgtagct gtagtagtag ctgtagtcga tcgacagtgt ccgagccaat 720

ccatcgccgc aaacaagcca aggggaagaa atatatatag ccctggccac gtactacgta 780

ctagcctctg ccgatctgcc atgggcgcta gtatagccat gtgaccgtgc gagagtgcaa 840

gtgatgggta cagacgttgt tagcaccaat ctgggaggct tatctccaaa cccccaaatt 900

ctctccattc cattgccgtc cgtcgtgtgg ctgccgcacg ctggaggggc agcggcaatc 960

gctagatta cgagggtccc acggcgcagt ggcacgacga cgcagcaccg tccggggattt 1020

tcaacggcac ccgaacctgg gcgtatcaat cgctttgtct cccggaaaag agaacgacat 1080

gggatcgtat taccccaagc gtcgccgatg atcgccaaaa gtcaaaaaaa cgtatcaccg 1140

tccaccgacc gaaccgaggt cgctaccacg tactaccagt agtagtagta gtagtacgcg 1200

gactcgcgac cagccagccg ctcgggttgt ctcctctttt ttttttttct tgccgggatg 1260

ggagcgtgca ccggtggctg cactcaccgt caccggccag tagtctaggc gacacacgaa 1320

gcggcggcct gcgtcgccga aaggcggggg aaaacccccga aaatacagga gaattctgcg 1380

tttcccgcgg gcgcgagagc ccgcgacccc gcgcacggtg agacagggac cacgaccccg 1440

cgcgaactcc cccttcaggt ggacgaccaa cccagcccag ccagccgcgc gggggtttgt 1500

agctttgccg aatccaacgc ttgcacacac caccgcggcg ccgtatgccg ggtcccccac 1560

ttgcattcca cccggtcgtc gcgtcgcgtg atcgcgtcac gtcccggcgc gccaggatac 1620

gggtgcttgg ctccgcctcc ggcttattat ttaagcacgc gacgccgcgg ccatgtctag 1680

tctcctcggc tgctactact actgcgttgt gcgcacaatg ccaagcgacc cactactgcc 1740

ctgcgtctgc gtgtgccgcg tatccgcaca agccaaagag aatttttagg cgggtagaaa 1800

caaaaatcag accccagtttc gcgcaaaaaa aaaacaaaga gaagcccggg ggtaagagag 1860

agagagagaa tttgagagat gtgagacgga agcaacaagc acatgagcgt tttgtgatta 1920

ggaacggaca aaacaaggtc ataaggcggc ctaatcagga ttggaggaga ggtctagtct 1980

ccctttctag tttcccctac atgaccagcg ttgccaccag gattgccacg ctcgtgcggg 2040

ccgcggcggc ggcgagccgg ccattgcggc tccaccgccg gcccggcggc gaggatacga 2100

ggatggtggt gatcgtcggc gccacgggca ccgggaagac caagctgtcc atcgacgccg 2160

ccaaggtgat cggcggcgag gttgtcaacg ccgacaagat tcagctctat gacggcctcg 2220

acgtgaccac caacaaggtg agcctcgccg accgccgcgg cgtgccgcac cacctcctcg 2280

gagccatccg ccccgaggcc ggcgagctcc cgccgtcgtc cttccggtcc ctcgccgccg 2340

ccacggccgc gtcgatcgcg gcgaggcggc tcgtgccggt catcgccggt gggtcgaact 2400

ccctcatcca cgccctcctc gccgaccact tcgacgcctc cgctggcgat cccttctccc 2460

ccgccgccgc cttccgccac taccgcccgg cgctccggtt cccgtgctgc ctgctctggg 2520

tccacgtcga tgaggcgctc ctcgacgagt acctcgaccg ccgcgtggac gacatggtgg 2580

acgctggcat ggtcgaggag ctccgggagt acttcgccac gacaaccgcc gcggagcgcg 2640

ccgcgcactc cgggctgggc aaggccatcg gcgtccccga gctcggcgac tacttcgccg 2700

ggcgcaagac cttctccgag gcgatcgacg acatcaaagc caacacccgc gtcctcgccg 2760

ccgcgcaggt gtccaagatc cgccgcatgt ccgacgcctg gggctggccc atccaccgcc 2820

tcgacgcctc cgacacagtc cgcgccaggc tcacgcgggc gggctccgcc gccgagtccg 2880

cctcctggga gcgcgacgtg cgcggcccag gcctcgccac catccgcagc ttcctcgccg 2940

atcagtcacc gccaccgcgc agcgagggca ccaacgacta cctgtacgcc atggagacgg 3000

aaccagagcc gccgccgccg ccgacgttgc cgccgcggct gctccggttg ccgcggatgc 3060

agtactgcga catggtgggg tgagctcgcc gccgccgccg ccgccgccgc acggcgccgc 3120

agtcacttca ttgaaggctt ttgggggttc gagggtttag gccgccaatt ttccagtgtc 3180

ccggcggcgc ccttctctga cactgctcgg tgggcccaga aaaagcagcc agtgacatag 3240

aaagaagaga caaaggtaat taacgtagag agagaagc taagctatgc aactgatgag 3300

aagtgttctt cttcattgca gcagaatttg tgtactcttt cttggagagg tggtgattca 3360

tcacatcgct ctcctcctaa ccgcggcact gtatgtatgt agtgagtagt actaatcatc 3420

taattatcca ccgtgttcag aattttgaga cagataatga gtacagtcta gtatatgatt 3480

tttcatcagt gtttgttgca gtgcaacggg tgacctgctc tatttccgga gctgaatttt 3540

ttttggtttg gttttgtttg ttttgtttgt ttaattaatc ctcctcctcc tcctgctgct 3600

gctgctagta gtagtagttt gaggtggcta atgccatggt ggatgcttga catgttccat 3660

ccattttcca gctggttgtt gttgggatga ttggatgggt aatgccacgc ttagtgtgtg 3720

atttctgatc gagggggaga aggattgtgt tgggcgtgtg ggttggtgcc tggtgttctt 3780

gctgttgcca gctaggaaca aacaaattcg tccccgtctc ctactctttt ttctttcttt 3840

ctttctttct ttctttcttt ctttctgttt tttttttctt ttacccctcc tctccagttt 3900

tcccgtacgg tcaaagagct ggaattcagt ggcattacga ttttcactct cttttggatt 3960

gttttctttc ttgtttggat tctacagcag cagtctaccg catcagattt tcagcatcgt 4020

tttatactgt cgcggacttc aggcattgga tactcgcttc cagaagtttc tggtcatttt 4080

tttctttttc tttttcaaaa aaaattgcac ttcacatggt cggttggtac gcgtatgtgt 4140

acgttgtatg cagagttgta cattgtttta tgcgtatgta taccttgtag gcgacacgtc 4200

cgacataagc caggcttttt atacactgtc atcgatcacc tgccctcaaa tcgccatgat 4260

tagcagcaat gcaagtactg tgttttgctc gaagcggttc tactgtaata taatgtcgca 4320

gtactatacc actgctctat ggtactgtac tccctatttc agtatactcc agtccagtac 4380

ttttgcctgt accatcgctg cagtgtacga atgccaagtc tcctggataa tcgcaagtaa 4440

gcagattctt ggggacggct cggcccaaat agaatgcagc atcgcaaccc tgaagaagcc 4500

cagcaactgc cgtcaagctg tcatacggct atttcactac tcctttcatt tcatcctaaa 4560

atatactccc tccgtttcac aatgtaagtt attctagcat tttccacatt tatattgatg 4620

ttagaaagtg aaacggagga agtaaagaat tatacccttt aaatttatat gaaaatataa 4680

gtaattttgt aatcccaaga cagtacttta ttcctcactt atcatctttt atatagttta 4740

tttttcactt atcacctttt gtattgatgg atttcccgtc taatcacttt tttactactt 4800

tgttcttcca accatttgta tcgtgatttg ttcttttact attatcttaa tttttgtaaa 4860

aacaaatagt agaatccctt atattcacgg acggaaggag tacttcctta ctagtcagaa 4920

gggattgaac cggactacta gttgtggtta acaacagaat gatagacctg cgtttgggct 4980

ccgtcatcat tagctttttg catttttact tctgcttatt gaaatataac tggccaattt 5040

agtttgaaaa agaaaaaaga actgacgagc aaatgcaccg ttatttgagc caatagactg 5100

acagagtatc agacagttta agtgtgctgg gccgcgtcag ccttggccaa caatttcata 5160

tcctctgatc gtctcatcca atagtaggaa aggatgttca caagagtgtt caagctacgc 5220

tctaacttta ccacatgaag gattcagata tcagaactcg tcactgtttc ttgacatgat 5280

caaatttcgt ttgtaaaact gtgcaatatc acttccaggc tttgcctccg gcctattttg 5340

caggattgct gtactggatg agacagtcaa ggtatgggcc agactaacga aagggcctct 5400

cagtctcatc cccagccttt catgtactgc aatgtggaac atcaactgta attacagaac 5460

acttgttgta catctgaggg aatgttttgt gctgttcttc ccaatccaat tgcaggagtt 5520

agatatttgg aagcttaaga tagccaatgg atttctaaaa attacaatgt gtctgaaaga 5580

ttggtcaggc tatccccttg gagtgtgatc ttgatgtcgt gtgctgcatg ccacacgttc 5640

agagaaacat tccccacgtt ggattttttg aaggttctca catcaccaaa tgaggcattc 5700

agaaagtaca gtaaagtcac atctcgagga tgacaaaaga cgttcaaagg ataattgggt 5760

agggagttgg cacaaatgta cactatctca tcaacgcagt tctgcactag tttgaacaaa 5820

cacactctct tgtgcgatta caacaccagc gacatgtcca tgaaaaagaa gagaacggac 5880

acgtccaaaa ggcactcaac aatattgctt tcgccaaaat tcatatgaat taatccagca 5940

gatattttaa acatgatcag tatataggta tgctaataat tactagtttc agttagtcag 6000

tagccacaag aatctggact atggagaact ggaacggctg gaagggcgca ctgatcatgc 6060

atcaagccac tcaagagtga agaacatcat ctagctctag ccaaacggca ctacctgtca 6120

ggaacaaagc ctggagatac tgatggttca gccaaaaaag ataagcattt tacctgaaaa 6180

ataactcata aacctaacat ggaatttctg aaatttgatg tccacaggac cacagcagaa 6240

gtaaaatgac tacttgtacc accattctgc gtctgcaaga gtaaaacatt gacactacag 6300

gctggatgga gacccgcatc agacatataa tgcaaaccac tggaaccaaa tcaaccaatt 6360

atgtgcaaca tgtaaaattg taaatcgtga acatgaagca ctattaagtt ttaactcttg 6420

atttacaaaa taaaaaatca atcataggaa atgatcagac agacaaaatt gaagtcccag 6480

ggcatactgt caatcaaata atccaataac accagctccg aacacaaccg tgcataactt 6540

aaaaacctga ttaccgactc accatggatg tcatcagaat actaaattag tgcataatta 6600

gattgctaag tgagtatgga aaaaggacac aatgcaacaa tcaagcagtc tttcagctcc 6660

tgcatagtgc acacaggtac acaactggtg ttccatatgt caagaacgat aagcagaagt 6720

tcaaattatt aatgcactta gcaaatcgtt ctaataagca gtgtcaggat atgactagtg 6780

aacacttcgt ctcctccaaa agtgcaaaac agagatcaga cgaaaaacag aagtatcagc 6840

attcgagaag caagaatgat gtgcaccagc ttttgcaaac aacagaatat tgcaaaatga 6900

acaaatatat tgaacaaagg tatatgtaaa aatgaacaac taattctgga agaggtaatt 6960

aatgccagct aagacctttc ttaattgaaa attggcaatg attttgccgt tctgttaaaa 7020

aaaattaatg cgagctatac ttacaagcag gagattataa caaagcgcac ccttgataac 7080

tagtttgatt tcctacctga atttcactag ctgagatttc tttttctctt ttcaaaccac 7140

tttgttgcac cccaagtaca ctttcatttt cttcggtatc aatgtccaac tcaagacatg 7200

atatatgccc tgcagagaaa ccaaagctgc atctttttcc ctctatctac aatgcagaaa 7260

gtagagctta atacaaggaa atatcatcct cactaacact acaaaaccac atagtgatat 7320

catagccaac aacttgcaga gttgaagcga tttgcaccga aagaaatatt tcacagactg 7380

aaaccatgta tataacatga cagctaacca aacatcacac cacatatgcc acagggcaca 7440

aacatcgcat acgattccac ctttcttcta attagaaaac ctattcacaa tcaaccataa 7500

aatctatatc catcctctga tctagaagaa ccaacataat tattggatgg acatgttagc 7560

cgtcagaaga aaaaaaagca aaacatggga acatctgaaa agttgctc 7608

<210>61

<211>455

<212>PRT

<213> Rice

<220>

<221>CHAIN

<222>(0)...(0)

<223>OsIPT9 amino acidi (005021_3)

<400>61

Met Thr Ser Val Ala Thr Arg Ile Ala Thr Leu Val Arg Ala Ala Ala

1 5 10 15

Ala Ala Ser Arg Pro Leu Arg Leu His Arg Arg Pro Gly Gly Glu Asp

20 25 30

Thr Arg Met Val Val Ile Val Gly Ala Thr Gly Thr Gly Lys Thr Lys

35 40 45

Leu Ser Ile Asp Ala Ala Lys Val Ile Gly Gly Glu Val Val Asn Ala

50 55 60

Asp Lys Ile Gln Leu Tyr Asp Gly Leu Asp Val Thr Thr Asn Lys Val

65 70 75 80

Ser Leu Ala Asp Arg Arg Gly Val Pro His His Leu Leu Gly Ala Ile

85 90 95

Arg Pro Glu Ala Gly Glu Leu Pro Pro Pro Ser Ser Phe Arg Ser Leu Ala

100 105 110

Ala Ala Thr Ala Ala Ser Ile Ala Ala Arg Arg Leu Val Pro Val Ile

115 120 125

Ala Gly Gly Ser Asn Ser Leu Ile His Ala Leu Leu Ala Asp His Phe

130 135 140

Asp Ala Ser Ala Ala Gly Asp Pro Phe Ser Pro Ala Ala Ala Phe Arg His

145 150 155 160

Tyr Arg Pro Ala Leu Arg Phe Pro Cys Cys Leu Leu Trp Val His Val

165 170 175

Asp Glu Ala Leu Leu Asp Glu Tyr Leu Asp Arg Arg Val Asp Asp Met

l80 185 190

Val Asp Ala Gly Met Val Glu Glu Leu Arg Glu Tyr Phe Ala Thr Thr

195 200 205

Thr Ala Ala Glu Arg Ala Ala His Ser Gly Leu Gly Lys Ala Ile Gly

210 215 220

Val Pro Glu Leu Gly Asp Tyr Phe Ala Gly Arg Lys Thr Phe Ser Glu

225 230 235 240

Ala Ile Asp Asp Ile Lys Ala Asn Thr Arg Val Leu Ala Ala Ala Gln

245 250 255

Val Ser Lys Ile Arg Arg Met Ser Asp Ala Trp Gly Trp Pro Ile His

260 265 270

Arg Leu Asp Ala Ser Asp Thr Val Arg Ala Arg Leu Thr Arg Ala Gly

275 280 285

Ser Ala Ala Glu Ser Ala Ser Trp Glu Arg Asp Val Arg Gly Pro Gly

290 295 300

Leu Ala Thr Ile Arg Ser Phe Leu Ala Asp Gln Ser Pro Pro Pro Arg

305 310 315 320

Ser Glu Gly Thr Asn Asp Tyr Leu Tyr Ala Met Glu Thr Glu Pro Glu

325 330 335

Pro Pro Pro Pro Pro Thr Leu Pro Pro Arg Leu Leu Arg Leu Pro Arg

340 345 350

Met Gln Tyr Cys Asp Met Val Gly Arg Ile Cys Val Leu Phe Leu Gly

355 360 365

Glu Val Val Ile His His Ile Ala Leu Leu Leu Thr Ala Ala Leu Ile

370 375 380

Leu Gln Ser Ser Thr Phe Ala Cys Thr Ile Ala Ala Val Tyr Glu Cys

385 390 395 400

Gln Val Ser Trp Ile Ile Ala Ser Phe Ala Ser Gly Leu Phe Cys Arg

405 410 415

Ile Ala Val Leu Asp Glu Thr Val Lys Glu Leu Asp Ile Trp Lys Leu

420 425 430

Lys Ile Ala Asn Gly Phe Leu Lys Ile Thr Met Cys Leu Lys Asp Trp

435 440 445

Ser Gly Tyr Pro Leu Gly Val

450 455

<210>62

<211>5075

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223>OsIPT3 genome sequence (002374_2)

<221> exons

<222>(2001)...(2503)

<221> exons

<222>(2544)...(3075)

<400>62

atataaaaaa cgaaaaattg tacttaaagt actttggata ataaagtaag tcaaaaaaat 60

aataattcta ttttttaaat aagacgagtg gtcaaacagt gaaaaaaaac tcaaaatccg 120

ttatattacg gaacggagga agtagcttac acggtgtagt acagtggcag cagtacgtcg 180

tctgttggct ctggtcaact gtaccgttat atataaagtg tcactcactt tgagagaaaa 240

agaggcatca tgacacgggc accaccartt gtatggatac gtaactacca tccatatata 300

tgcatatcag tgtcgaatgc aatcagctca tctcgatcca gtctccaatt tcaaaagaga 360

agatgtttga aattttaaca ttttagtttc ttttttgaaa atttactaca caaatatact 420

tcttaaaaaa actaattcta tatacctcgc ctcaaaaaaa atctctaaat tgatttgtct 480

gctcaagcta gtctcttttt aaaaaatgaa taagacaact ccattgttca gatagcccaa 540

cacagttgaa ctgctagcta ctccctccat cccataatat aagggatttt gagtttttac 600

ttgtaacgtt tgatcactcg tcttattcaa atttttttaa aactattatt tattttattt 660

gtgacttact ttattatcta cagaacttta agcacaactt ttcgtctttt atatttgaaa 720

aaaaatttga atgagacgag tggtcaaacg ttgcaatcaa aaactcaaaa tcccttatat 780

tgtgggacgg agggagaacc aaattacaaa caatgtctgg gaatccttca agccaaacaa 840

agtgactttg tagacctaga gatgcgtcaa ttttagcaag ctcatgggta acacgattat 900

gttctctagg acaattcata acttgataga caaaaaaaaa tcattgtaag cgcataaatt 960

tggcctatct aatgataaca acttttactc ttgatcagac agcaagagtt tttttttatt 1020

gacaaaattt aggcatatta tctgaaaacc ccaggtggcc gcgtgtggcc gcgcgcgtcc 1080

gcggcgtgct tcttctgaat gctcacaatg acgccatcct catcagccgc tgcggagcac 1140

tccaccaagg tcagcatcct cgacgcaccg gcgcagcgta cgtctcatgc atgcatcctt 1200

tatcgtacaa gaactattac tccatccggt ttcatattcc taacatttta aacaaggtta 1260

aggacgttaa ggtctctttt agaattttgc actatcaata actacaccag cggcatacat 1320

agattactct taaaaagcac tatcaataaa gtaaatattt atttattcac tgtatatata 1380

ttataataga aaactataac taaagaaatg ttttggcgat cgtgcatgtg gaaaatataa 1440

tttgttgtat tattgtagtg actctctcac agatatatat agagtatatg tgagatagag 1500

atttagagta caatacaaat tttaaaacag atttatttct agaactacat ttatctataa 1560

taatattcta tctctagaat tctatcttat ctctataatt tatatttaaa ctttcctccg 1620

aacatttatt ttaagaagaa atttatagcg tattgggaat tgaacgcggg atttttaggg 1680

ttgaaaccac atacctcttg tcactgcact atcaaatgca tctcagagca ctgcagcatg 1740

atcactaaac tccctctccc cctaacgact gcaaggcgcc gcgtcctcca accccacagtg 1800

tgggtgatcg cccttcacat tccagagcgc cctctccccc cctataaata ccccactgcc 1860

agcctcatct tcctccacag catccatcgc aaaatccacc tcagctagct acccaagcgc 1920

acgcgccacc gcccgcgcgc gagctgagct aacgatcaac accggtcgaa cacctagcga 1980

gcatcaccac catcatcacc atgcaggcgt acatggcggt cgccgccgct ccggcgccac 2040

cggcctcgct gacgctgctg ccgcgcacca ccaccgtcat cagggacagg gagcgcttcg 2100

acgcggccgt cccggtggcg ccgctcgtgc tgaggcatgg cgccggcgtc aagcacaagg 2160

ccgtcgtggt gatgggcgcc acgggcaccg ggaagtcacg cctcgccgtc gacctcgccc 2220

tgcggttcgg cggcgaggtc atcaactccg acaagatgca gatacattcc gggttggatg 2280

tggtgacgaa caaggtgacc gaggaggagt gcgccggcgt gccgcaccac ctgatcggcg 2340

tggcgcgccc cgacgacgag ttcacggccg ccgacttccg ccgcgaggcg gcgcgcgccg 2400

cggcgggggc ggttgagagg gggaggttgc ccatcatcgc cggggggtcc aactcctacg 2460

tcgaggagct cgtcgaaggc gacggccgcg cgttccggga gcggtacgag tgctgcttcc 2520

tctgggtcga cgtggatctc gaggtgctcc gcggtttcgt cgcccgccgc gtcgacgaga 2580

tgtgccggcg aggcctcgtc cgggaggtgg ccgcagcgtt cgacccgcgc cgcaccgact 2640

actcccaggg gcatctggcg cgccatcggc gtgccggagc tcgacgcgta cctccgctcc 2700

cgcggcgacg gcgccgacga ggaggagcgg gcgcgcatgc tcgccgcggc cgtcgcggag 2760

atcaagtcga acacgttccg gctcgcgtgc cgccagcacc gcaagatcga gaggctggac 2820

cgcatgtggc gcgcccgccg cgtcgacgcc acggaggtgt tcaggaggcg cggccacgcc 2880

gccgacgacg cgtggcagcg gctcgtcgcc gcgccgtgca tcgacgccgt ccggtcattc 2940

ctcttcgagg accaagaacg cagcagcatc gccgccggca aacctcccct cttcgccgcc 3000

ggcaaggcca cttcaggcaa catctccgtc ttcgcctccg cggccgccgc catggcggcg 3060

gccgctgcaa tctgagaagc gcaggcacca acctacttac atacacatac atgaccaaac 3120

acaaaaacgc agagcaccat gccactctca agacattcag gctgctgcca atcgccattg 3180

ccgatcaaat tcagagctcg tcagtcggat caaacgatga aggctgctgc ttctgcggtc 3240

aacatctcga tgacctgcca attgcaatgc aaagcaagat tgttcatata ctggtaactg 3300

gttgactcct ttttgttttt gctgggtttc tttgtgatat gaggagattg atgaattgcg 3360

cgcgagaatg taacttatga gttttgagtt tttttttctt tttggtttct cctctgattt 3420

tgggtgtgta acagtagggag taaaaactaa aaagtattga cttggatgtt aattggggga 3480

ttgatgtaat agcctctcct tgtaacatag aaatcatgag ctgaaataat taaatcgttg 3540

agaccttgca aagaaaaagg gggaaaaaca aaagaaggaa tcttgtgaaa ggggaatatt 3600

tggcagtctt tatcgtaata tatcaacttt ttcaaggtct tcactgagct ttgctattga 3660

gaatctcact tgccgcgcac ttcaatagtc tatgactatg ctctacactg tccttgttct 3720

taattcgaga ccgcatactt tgagtgacat gttgtgggta tgagtaaaac tcggtggcac 3780

gcaatactaa tttttcttta catgaaaact tataattcgc acaattttgt gtcaaaaaaa 3840

aattcgcgca atccttttgc gcattcccta taaaaactta taaattgtct gtatttataa 3900

tttttatttt cttacgttct ctatttcatt cagaaaactc ctggccaatc accatgacag 3960

gtgatacatg caacatttgg cctggaaaac tagtggcagt cagccagtca ttcctcttgt 4020

gcaatgtgtg gctctatgct gtatctgtct gtggatacaa gttggcatct gcatcagttc 4080

tggttgcttc tttgacatca tattcccaag tcttgtttct tggtcatgga gacactctca 4140

tgcatgattg ggaactgggt attagttagg gcagtgcttg catgtcaaag gtcaagccca 4200

tcatcttctt gttcaaagtc aacttcacat tctgaatgca tatgcacatt tttacaagga 4260

taagcttgat ttaacacata ttttagagtt cacttgaaca ttgtcaaact ctttcgtaga 4320

atgcagtggt gatctctctg gatagtatgg agaccagaag actcagcgat catcggttca 4380

gaatattata taagattcag aaggtcaaca tgaacaaata tttatatgaa atagtaaaat 4440

tggtcaggta aatgataact ttgacttaat cctactacag ttagacagtc aaaatggcat 4500

ttcttgatgc tacatttttt ttctacatgg attgacaaaa acgaatgaac gaatcagagt 4560

tatttgacca atttatcaaa ttatgtcaag tggatgatca gagaataaga tctgaatatc 4620

gcgataggaa tgaaatcttt ggtcaagggt caatgtgcag ttcctgcatt tccttcccca 4680

aatcaaacct agaactggtt gacacttgac agagaatgca acgactctgt ttttgttttc 4740

atttttttgg aagattcttc tatgaacact ggctctttga ccaactgcct acatatctct 4800

actgatacat gagtgatcag taataatttc tgttgtacaa tgttttctat agcattctga 4860

tttgccaata ataattaaca aagtcaaaag cttttttttt tctgaaggag aaagtcaaac 4920

tgttcttctc aaatacagag tccaacttgc aacatggtga acaaaacaaa aggaggaaaa 4980

tacgaaagga atcagcaaat agctaacaag ctgatcattg agcaatgaga aaaatatcac 5040

atttaataaa ggtttaaagt caacactaga gcttg 5075

<210>63

<211>344

<212>PRT

<213> Rice

<220>

<221>CHAIN

<222>(0)...(0)

<223> OsIPT3 amino acid sequence (002374_2)

<400>63

Met Gln Ala Tyr Met Ala Val Ala Ala Ala Pro Ala Pro Pro Ala Ser

1 5 10 15

Leu Thr Leu Leu Pro Arg Thr Thr Thr Val Ile Arg Asp Arg Glu Arg

20 25 30

Phe Asp Ala Ala Val Pro Val Ala Pro Leu Val Leu Arg His Gly Ala

35 40 45

Gly Val Lys His Lys Ala Val Val Val Met Gly Ala Thr Gly Thr Gly

50 55 60

Lys Ser Arg Leu Ala Val Asp Leu Ala Leu Arg Phe Gly Gly Glu Val

65 70 75 80

Ile Asn Ser Asp Lys Met Gln Ile His Ser Gly Leu Asp Val Val Thr

85 90 95

Asn Lys Val Thr Glu Glu Glu Cys Ala Gly Val Pro His His Leu Ile

100 105 110

Gly Val Ala Arg Pro Asp Asp Glu Phe Thr Ala Ala Asp Phe Arg Arg

115 120 125

Glu Ala Ala Arg Ala Ala Ala Gly Ala Val Glu Arg Gly Arg Leu Pro

130 135 140

Ile Ile Ala Gly Gly Ser Asn Ser Tyr Val Glu Glu Leu Val Glu Gly

145 150 155 160

Asp Gly Arg Ala Phe Arg Glu Arg Cys Ser Ala Val Ser Ser Pro Ala

165 170 175

Ala Ser Thr Arg Cys Ala Gly Glu Ala Ser Ser Gly Arg Trp Pro Gln

180 185 190

Arg Ser Thr Arg Ala Ala Pro Thr Thr Pro Arg Gly Ile Trp Arg Ala

195 200 205

Ile Gly Val Pro Glu Leu Asp Ala Tyr Leu Arg Ser Arg Gly Asp Gly

210 215 220

Ala Asp Glu Glu Glu Arg Ala Arg Met Leu Ala Ala Ala Val Ala Glu

225 230 235 240

Ile Lys Ser Asn Thr Phe Arg Leu Ala Cys Arg Gln His Arg Lys Ile

245 250 255

Glu Arg Leu Asp Arg Met Trp Arg Ala Arg Arg Val Asp Ala Thr Glu

260 265 270

Val Phe Arg Arg Arg Gly His Ala Ala Asp Asp Ala Trp Gln Arg Leu

275 280 285

Val Ala Ala Pro Cys Ile Asp Ala Val Arg Ser Phe Leu Phe Glu Asp

290 295 300

Gln Glu Arg Ser Ser Ile Ala Ala Gly Lys Pro Pro Leu Phe Ala Ala

305 310 315 320

Gly Lys Ala Thr Ser Gly Asn Ile Ser Val Phe Ala Ser Ala Ala Ala

325 330 335

Ala Met Ala Ala Ala Ala Ala Ile

340

<210>64

<211>4777

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT4 genome sequence (000911_6)

<221> CDS

<222>(1734)...(2778)

<400>64

gaacagagat taagtgatta acacatacga gaggcaagat gatggacaca cccacaggat 60

cgcctgccga tcgatggcac gccgtccgac caggtcgatg gagacccaga agccggccaa 120

caactaatgc catagaaaaa ttttacagcc tagagacaaa catatttgaa aaaattttag 180

atatatattt tcaaaccata gagtttcccg gatggttatg gatccacccc tggcaacagg 240

acaagtcatc tagatggaga gtaaaaagtc gcgcgaggac gaagctgatg aacctcgtct 300

ttttttttct taacctgaaa aacaactagt atgtacaact aaagttgcat ttagggttat 360

tatatccttg ttagtattga tttcaaatga aagttatcag tggtaaataa gaaaaagttg 420

tacaagaata tctggaatgg cttcattgcc tttaataatt gacaatgaca atcaagaaaa 480

gaaaaaaaaa gataacattt ctaggtgttc acaaactcac gaagagtgtc gacaagtttg 540

caacaacatg tatttgaaat cggagaaagt actctctcta tactaaaata attttttttt 600

aaattttatc atttgttcta aaataaattt acttttatca ccttatttac cctaattgat 660

gcaccaattg aaagcttatt atttaaatat ctcttaccta cctatccact aatatactat 720

actttcttat taactagata atgtatcgca ctttgctatg agatatatgt tagttatga 780

agatacaata aaataatttg gattgaaata ttatgaaaat gatttgagaa tgatgatta 840

gcatgtgtat gtttagtttt agaatgaaat aagttgtaga tataattact acgtgcttgc 900

atgttgaact ttgtgtgttt tataggttga tgtggcatgc ttgcatgcag attttaggag 960

tgctaataaa tactccctat atttgcatgt tgagttttag gtgtttagtg gatattagct 1020

ttatacaaag aagagataaa gagcacttcg tttttttcct ctgataattt gaaatccatt 1080

ggctaaagat gaagttatt tagatggagg tagtactttt tttatctgat agtgcactac 1140

atctctctac cttgtgcatt tgttttcatt cttgtttttc attcttgtgg tcctctacat 1200

aaaagatgtt acgtagtctc tatcgacact aaggatttct ttggagaagc ttctagcagc 1260

tacaactata ttaaaaatct ctaacttttt gctatatatt ttgaatctct agctccctaa 1320

gcagtataga ttctcgctcc aaattttaag agttagtaca tgtaaaattt agaaataaac 1380

tagtagctaa aagcagcgga gcttcgatct ctcaagattc tcataagtta tttcttagaa 1440

tctataattc cccaaatata gcatatatgt ttttagtaga tggctaaact tatactttat 1500

gcctcaaatt aaaactcgtt aattctgttg agtatctttg ttgtttgttc tccacttggt 1560

tttggttagc taactccata taaatacaca tccaaacaca cccacaagac agcaaccaac 1620

tccaagcttg ctaactaaca aagcatcttg aattcctctt gaccaagtag ttaagtagct 1680

aagctaatta gtggtctaac tttgaacaca acacaacaaa acacacacac atc atg 1736

Met

1

gcc acg tca cta tcc ttg gcg ccc aaa ccc gcc gcc gtc gcc gtc gcc 1784

Ala Thr Ser Leu Ser Leu Ala Pro Lys Pro Ala Ala Val Ala Val Ala

5 10 15

gcc gcc gcc atc ccg agg ctt gtt ccg ccg ccg tct atc gac atg tcg 1832

Ala Ala Ala Ile Pro Arg Leu Val Pro Pro Pro Ser Ile Asp Met Ser

20 25 30

gcg ctg tcg ccg ccg ccg ccg ctc gtc agc gtc agg agg agc atg gta 1880

Ala Leu Ser Pro Pro Pro Pro Leu Val Ser Val Ser Arg Ser Met Val

35 40 45

gcg aag cac aag gcc gtg gtt gtg atg ggc gcg acg ggg ag ag ggg aag 1928

Ala Lys His Lys Ala Val Val Val Met Gly Ala Thr Gly Thr Gly Lys

50 55 60 65

acg cgg ctc gcc gtc gac ctc gcg ctc cag ttc ggc ggc gag gtg atc 1976

Thr Arg Leu Ala Val Asp Leu Ala Leu Gln Phe Gly Gly Glu Val Ile

70 75 80

aac gcc gac aag ctg cag ctg cac cgg ggg ctc gac gtg gcc acc aac 2024

Asn Ala Asp Lys Leu Gln Leu His Arg Gly Leu Asp Val Ala Thr Asn

85 90 95

aag gcc acc gcc gac gag cgc gcc ggc gtg ccg cac cac ctg atc ggg 2072

Lys Ala Thr Ala Asp Glu Arg Ala Gly Val Pro His His Leu Ile Gly

100 105 110

gtg gcg cac ccg gac gag gag ttc acg gcc gcg gac ttc cgc cgc gcc 2120

Val Ala His Pro Asp Glu Glu Phe Thr Ala Ala Asp Phe Arg Arg Ala

115 120 125

gcg tcg cgc gcc gcc gcc gcg gtc gcc gcg cgc ggc gcg ctg ccc atc 2168

Ala Ser Arg Ala Ala Ala Ala Val Ala Ala Arg Gly Ala Leu Pro Ile

130 135 140 145

atc gcc ggc ggc tcc aac tcc tac atc gag gag ctc gtc gac ggc gac 2216

Ile Ala Gly Gly Ser Asn Ser Tyr Ile Glu Glu Leu Val Asp Gly Asp

150 155 160

cgc cgc gcg ttc cgc gac cgg tac gac tgc tgc ttc ctg tgg gtg gac 2264

Arg Arg Ala Phe Arg Asp Arg Tyr Asp Cys Cys Phe Leu Trp Val Asp

165 170 175

gtg cag ctc ccc gtg ctc cac ggc ttc gtc ggc cgc cgc gtc gac gac 2312

Val Gln Leu Pro Val Leu His Gly Phe Val Gly Arg Arg Val Asp Asp

180 185 190

atg tgc ggc cgc ggg atg gtc gcc gag atc gag gcg gcg ttc gac ccg 2360

Met Cys Gly Arg Gly Met Val Ala Glu Ile Glu Ala Ala Phe Asp Pro

195 200 205

gac cgc acc gac tac tcc cgc ggc gtc tgg cgc gcc atc ggc gtg ccg 2408

Asp Arg Thr Asp Tyr Ser Arg Gly Val Trp Arg Ala Ile Gly Val Pro

210 215 220 225

gag ctc gac gcg tac ctc cgc tcg tgc gcc gcc gcc ggc ggc gag gag 2456

Glu Leu Asp Ala Tyr Leu Arg Ser Cys Ala Ala Ala Gly Gly Glu Glu

230 235 240

gaa cgc gcg cgg ctg ctg gcc aat gcc atc gag gac atc aag gcg aac 2504

Glu Arg Ala Arg Leu Leu Ala Asn Ala Ile Glu Asp Ile Lys Ala Asn

245 250 255

acc cgc tgg ctg tcg tgc cgg cag cgc gcc aag atc gtg agg cta gac 2552

Thr Arg Trp Leu Ser Cys Arg Gln Arg Ala Lys Ile Val Arg Leu Asp

260 265 270

cgc cta tgg cga atc cgc cgc gtg gac gcc acg gag gcg ttc cgg cgg 2600

Arg Leu Trp Arg Ile Arg Arg Val Asp Ala Thr Glu Ala Phe Arg Arg

275 280 285

cgc ggc ggc gcc gcc aac gag gcg tgg gag cgg cac gtc gcc gcg ccg 2648

Arg Gly Gly Ala Ala Asn Glu Ala Trp Glu Arg His Val Ala Ala Pro

290 295 300 305

agc att gac acc gtg cga tcc ttc ctc cac ggc gaa ttc acc acc gcc 2696

Ser Ile Asp Thr Val Arg Ser Phe Leu His Gly Glu Phe Thr Thr Ala

310 315 320

gcc gaa act acg gcg gcg ccg gtg ccg cca ccg ccg ttc ctc ccc atg 2744

Ala Glu Thr Thr Ala Ala Pro Val Pro Pro Pro Pro Phe Leu Pro Met

325 330 335

ttt gct ctc gcc gcg gcg ggc gcc ggc gtc taa g ctcagctcag 2788

Phe Ala Leu Ala Ala Ala Gly Ala Gly Val *

340 345

ctcgaaacga cagtaaaaat tttaaaaaac ttgcagagat ggatggggag cttaataagt 2848

gtgaagtgaa ggtaattaag aagaaattaa ccactgtttg atgtaatgac attgatattg 2908

accaggccaa aaaagggaga gaaaaagagc agcagatgtg gaggagtgta ccatctgtgg 2968

actgagagtg acccttccga ttagggatgg aattggtcg atccacaaga acttcttgac 3028

ccactctgat tcagctaaat gaactaattt atacgattgg agttttgaaa ataaatttag 3088

ttcatatagc taaactagga tggttaagga gtattcgtgg gtcggcccac tctaacccct 3148

agctactttg gatcacagga ttttatggcg gtattttcac tttacaaatt agtatgagat 3208

ctttataagg ttgttgtgca ttgcatatgt aaatgcgatg gtctagtagg gtatgaatgt 3268

cgggcctctg tatgattctg ttgagagtga ttaatattag tttttctttg ttatttattt 3328

gaactacaat aaaaaaatac ggttgtgtga ttgagctagt gttacccaac cttcacctta 3388

attctgctta ttctggattg tttattcatc ttacttggaa atattaattt gtgagtgaga 3448

tgatctgatg atgaatgtaa taattatttg aggtgattga gtattgatct tgttatctat 3508

atattgtgtt ttgtattatc agattataca actagctagt tttttttcaa ttgatcgaga 3568

tatataactc ctatagctag ttgacctata tgggaagcta ggctagctag cttgtctgta 3628

ttgctaacat aaatgtgaat acatgataat tacatgttga ttgaaataac tctcagataa 3688

agaaactaat taaataagct agtagtgcac atgattttga taatgaaggc aaatttggct 3748

aatgtgcact gcgctggttg atattttctg atagtaatca agaatatatt ggtgctagtg 3808

ggcaccatga atttgtgcca gttgctaaca caaatggatc gaggccagct aacaagaata 3868

tataaaacac ctgagcttca accttagtag catgcatgca gttgattaa ttatcatcat 3928

tgtaatatct tgtaatatga ccttttcaac ttaatcatga aaaaaaattc cttcaagaaa 3988

ctagtaagtg acatcgttgc acatctggag aagtccatcg atcaacttga agtgcattca 4048

cagttgcaca agctaaatct gaattatggt tcctaggcta atatataatt cgaaaatacc 4108

ttctttgaaa aaaaactaac acaacttttg gtaaatatga aaatgcatag catggtatta 4168

aaattttcag aatattcaat attaaattta taccacttct taaccaattt aattagttct 4228

gctggtttca tgaatatttt taggcatatt ctgagtggat aaaaatatta aaatctaggc 4288

agtgatataa aataatttat caataatatc gttcattaaa ttaataattt tgcagtgtgg 4348

aagaaggtga tgctgtaatg catgaggaaa aatgggtccc cctatcctcc ggtaagaaaa 4408

ttccttaaga taaagcatca tatataaata tgttgattta gaaaatataa taattttaaa 4468

ttgtagccaa ttatatacac atatccgata caaattctaa caataattat taggttggta 4528

aaatgaattc atcagacata tattagccgt cggacatatg ttgtggttag tttatgtttc 4588

agtaagcttt taaacaaaat aggcgaattc taagagattg catgaaaacc acctttttga 4648

aacaaagtaa agcctataaa ccaccttagc ccgatcgaca tgttctaatg aatgagctat 4708

ggctacaaat atataactgt tctattagga accatattct ggaaatatat taaattaagt 4768

tccctcttt 4777

<210>65

<211>1044

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT4 coding sequence (000911_6)

<221> CDS

<222>(1)...(1044)

<400>65

atg gcc acg tca cta tcc ttg gcg ccc aaa ccc gcc gcc gtc gcc gtc 48

Met Ala Thr Ser Leu Ser Leu Ala Pro Lys Pro Ala Ala Val Ala Val

1 5 10 15

gcc gcc gcc gcc atc ccg agg ctt gtt ccg ccg ccg tct atc gac atg 96

Ala Ala Ala Ala Ile Pro Arg Leu Val Pro Pro Pro Ser Ile Asp Met

20 25 30

tcg gcg ctg tcg ccg ccg ccg ccg ctc gtc agc gtc agc agg agc atg 144

Ser Ala Leu Ser Pro Pro Pro Pro Leu Val Ser Val Ser Arg Ser Met

35 40 45

gta gcg aag cac aag gcc gtg gtt gtg atg ggc gcg acg ggg acg ggg 192

Val Ala Lys His Lys Ala Val Val Val Met Gly Ala Thr Gly Thr Gly

50 55 60

aag acg cgg ctc gcc gtc gac ctc gcg ctc cag ttc ggc ggc gag gtg 240

Lys Thr Arg Leu Ala Val Asp Leu Ala Leu Gln Phe Gly Gly Glu Val

65 70 75 80

atc aac gcc gac aag ctg cag ctg cac cgg ggg ctc gac gtg gcc acc 288

Ile Asn Ala Asp Lys Leu Gln Leu His Arg Gly Leu Asp Val Ala Thr

85 90 95

aac aag gcc acc gcc gac gag cgc gcc ggc gtg ccg cac cac ctg atc 336

Asn Lys Ala Thr Ala Asp Glu Arg Ala Gly Val Pro His His Leu Ile

100 105 110

ggg gtg gcg cac ccg gac gag gag ttc acg gcc gcg gac ttc cgc cgc 384

Gly Val Ala His Pro Asp Glu Glu Phe Thr Ala Ala Asp Phe Arg Arg

115 120 125

gcc gcg tcg cgc gcc gcc gcc gcg gtc gcc gcg cgc ggc gcg ctg ccc 432

Ala Ala Ser Arg Ala Ala Ala Ala Val Ala Ala Arg Gly Ala Leu Pro

130 135 140

atc atc gcc ggc ggc tcc aac tcc tac atc gag gag ctc gtc gac ggc 480

Ile Ile Ala Gly Gly Ser Asn Ser Tyr Ile Glu Glu Leu Val Asp Gly

145 150 155 160

gac cgc cgc gcg ttc cgc gac cgg tac gac tgc tgc ttc ctg tgg gtg 528

Asp Arg Arg Ala Phe Arg Asp Arg Tyr Asp Cys Cys Phe Leu Trp Val

165 170 175

gac gtg cag ctc ccc gtg ctc cac ggc ttc gtc ggc cgc cgc gtc gac 576

Asp Val Gln Leu Pro Val Leu His Gly Phe Val Gly Arg Arg Val Asp

180 185 190

gac atg tgc ggc cgc ggg atg gtc gcc gag atc gag gcg gcg ttc gac 624

Asp Met Cys Gly Arg Gly Met Val Ala Glu Ile Glu Ala Ala Phe Asp

195 200 205

ccg gac cgc acc gac tac tcc cgc ggc gtc tgg cgc gcc atc ggc gtg 672

Pro Asp Arg Thr Asp Tyr Ser Arg Gly Val Trp Arg Ala Ile Gly Val

210 215 220

ccg gag ctc gac gcg tac ctc cgc tcg tgc gcc gcc gcc ggc ggc gag 720

Pro Glu Leu Asp Ala Tyr Leu Arg Ser Cys Ala Ala Ala Gly Gly Glu

225 230 235 240

gag gaa cgc gcg cgg ctg ctg gcc aat gcc atc gag gac atc aag gcg 768

Glu Glu Arg Ala Arg Leu Leu Ala Asn Ala Ile Glu Asp Ile Lys Ala

245 250 255

aac acc cgc tgg ctg tcg tgc cgg cag cgc gcc aag atc gtg agg cta 816

Asn Thr Arg Trp Leu Ser Cys Arg Gln Arg Ala Lys Ile Val Arg Leu

260 265 270

gac cgc cta tgg cga atc cgc cgc gtg gac gcc acg gag gcg ttc cgg 864

Asp Arg Leu Trp Arg Ile Arg Arg Val Asp Ala Thr Glu Ala Phe Arg

275 280 285

cgg cgc ggc ggc gcc gcc aac gag gcg tgg gag cgg cac gtc gcc gcg 912

Arg Arg Gly Gly Ala Ala Asn Glu Ala Trp Glu Arg His Val Ala Ala

290 295 300

ccg agc att gac acc gtg cga tcc ttc ctc cac ggc gaa ttc acc acc 960

Pro Ser Ile Asp Thr Val Arg Ser Phe Leu His Gly Glu Phe Thr Thr

305 310 315 320

gcc gcc gaa act acg gcg gcg ccg gtg ccg cca ccg ccg ttc ctc ccc 1008

Ala Ala Glu Thr Thr Ala Ala Pro Val Pro Pro Pro Pro Phe Leu Pro

325 330 335

atg ttt gct ctc gcc gcg gcg ggc gcc ggc gtc taa 1044

Met Phe Ala Leu Ala Ala Ala Gly Ala Gly Val *

340 345

<210>66

<211>347

<212>PRT

<213> Rice

<400>66

Met Ala Thr Ser Leu Ser Leu Ala Pro Lys Pro Ala Ala Val Ala Val

1 5 10 15

Ala Ala Ala Ala Ile Pro Arg Leu Val Pro Pro Pro Ser Ile Asp Met

20 25 30

Ser Ala Leu Ser Pro Pro Pro Pro Leu Val Ser Val Ser Arg Ser Met

35 40 45

Val Ala Lys His Lys Ala Val Val Val Met Gly Ala Thr Gly Thr Gly

50 55 60

Lys Thr Arg Leu Ala Val Asp Leu Ala Leu Gln Phe Gly Gly Glu Val

65 70 75 80

Ile Asn Ala Asp Lys Leu Gln Leu His Arg Gly Leu Asp Val Ala Thr

85 90 95

Asn Lys Ala Thr Ala Asp Glu Arg Ala Gly Val Pro His His Leu Ile

100 105 110

Gly Val Ala His Pro Asp Glu Glu Phe Thr Ala Ala Asp Phe Arg Arg

115 120 125

Ala Ala Ser Arg Ala Ala Ala Ala Val Ala Ala Arg Gly Ala Leu Pro

130 135 140

Ile Ile Ala Gly Gly Ser Asn Ser Tyr Ile Glu Glu Leu Val Asp Gly

145 150 155 160

Asp Arg Arg Ala Phe Arg Asp Arg Tyr Asp Cys Cys Phe Leu Trp Val

165 170 175

Asp Val Gln Leu Pro Val Leu His Gly Phe Val Gly Arg Arg Val Asp

180 185 190

Asp Met Cys Gly Arg Gly Met Val Ala Glu Ile Glu Ala Ala Phe Asp

195 200 205

Pro Asp Arg Thr Asp Tyr Ser Arg Gly Val Trp Arg Ala Ile Gly Val

210 215 220

Pro Glu Leu Asp Ala Tyr Leu Arg Ser Cys Ala Ala Ala Gly Gly Glu

225 230 235 240

Glu Glu Arg Ala Arg Leu Leu Ala Asn Ala Ile Glu Asp Ile Lys Ala

245 250 255

Asn Thr Arg Trp Leu Ser Cys Arg Gln Arg Ala Lys Ile Val Arg Leu

260 265 270

Asp Arg Leu Trp Arg Ile Arg Arg Val Asp Ala Thr Glu Ala Phe Arg

275 280 285

Arg Arg Gly Gly Ala Ala Asn Glu Ala Trp Glu Arg His Val Ala Ala

290 295 300

Pro Ser Ile Asp Thr Val Arg Ser Phe Leu His Gly Glu Phe Thr Thr

305 310 315 320

Ala Ala Glu Thr Thr Ala Ala Pro Val Pro Pro Pro Pro Phe Leu Pro

325 330 335

Met Phe Ala Leu Ala Ala Ala Gly Ala Gly Val

340 345

<210>67

<211>484

<212>PRT

<213> Artificial sequence

<220>

<223> consensus sequence

<221> variant

<222>3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

61, 62

<223> Xaa = any amino acid

<221> variant

<222>63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92,

114, 116, 117, 145, 148, 157, 159, 172, 173, 176, 180, 182,

183, 204, 205, 206, 207, 209, 210, 211, 212, 213, 214

<223> Xaa = any amino acid

<221> variant

<222>215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,

227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,

239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,

251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261

<223> Xaa = any amino acid

<221> variant

<222>262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,

274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,

286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,

298, 299, 300, 301, 302, 303, 304, 305, 306, 319, 320

<223> Xaa = any amino acid

<221> variant

<222> 321, 324, 325, 328, 333, 336, 339, 340, 347, 352, 353, 354,

356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 369,

383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,

395, 396, 397, 398, 399, 400, 401, 402, 403, 406, 409

<223> Xaa = any amino acid

<400>67

Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

65 70 75 80

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Lys Val Val Val

85 90 95

Ile Met Gly Ala Thr Gly Thr Gly Lys Ser Arg Leu Ser Ile Asp Leu

100 105 110

Ala Xaa Arg Xaa Xaa Phe Gly Gly Glu Val Ile Asn Ser Asp Lys Ile

115 120 125

Gln Val Tyr Ala Asp Gly Leu Asp Val Ala Thr Asn Lys Val Thr Leu

130 135 140

Xaa Glu Arg Xaa Gly Val Pro His His Leu Leu Gly Xaa Ile Xaa Pro

145 150 155 160

Glu Ala Gly Glu Leu Thr Ala Ser Asp Phe Arg Xaa Xaa Ala Ala Xaa

165 170 175

Ala Ile Ala Xaa Ile Xaa Xaa Ala Arg Gly Arg Leu Pro Ile Val Ala

180 185 190

Gly Gly Ser Asn Ser Tyr Ile His Ala Leu Leu Xaa Xaa Xaa Xaa Asp

195 200 205

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

210 215 220

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

225 230 235 240

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

245 250 255

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

260 265 270

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

275 280 285

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

290 295 300

Xaa Xaa Leu Arg Tyr Asp Cys Cys Phe Leu Trp Val Asp Val Xaa Xaa

305 310 315 320

Xaa Val Leu Xaa Xaa Tyr Leu Xaa Arg Arg Val Asp Xaa Met Val Xaa

325 330 335

Asp Ser Xaa Xaa Gly Leu Val Glu Glu Leu Xaa Glu Phe Phe Asp Xaa

340 345 350

Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Ile

355 360 365

Xaa Lys Ala Ile Gly Val Pro Glu Leu Asp Glu Tyr Phe Arg Xaa Xaa

370 375 380

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

385 390 395 400

Xaa Xaa Xaa Leu Asp Xaa Ala Val Xaa Glu Ile Lys Xaa Asn Thr Xaa

405 410 415

Xaa Leu Ala Xaa Arg Gln Val Xaa Lys Ile Xaa Arg Leu Xaa Xaa Xaa

420 425 430

Xaa Gly Trp Xaa Ile Xaa Xaa Arg Val Asp Ala Thr Glu Xaa Phe Xaa

435 440 445

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

450 455 460

Xaa Glu Xaa Trp Glu Arg Xaa Val Xaa Xaa Pro Xaa Val Xaa Xaa Val

465 470 475 480

Arg Xaa Phe Leu

<210>68

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> Consensus sequence of ATP binding domain

<221> variant

<222>(1)...(1)

<223> The first amino acid can be G

<221> variant

<222>(8)...(8)

<223> The amino acid at position 8 can be T

<221> variant

<222>(2)...(5)

<223> The amino acid at position 2-5 can be any amino acid

<221> variant

<222>2,3,4,5

<223> Xaa = any amino acid

<400>68

Ala Xaa Xaa Xaa Xaa Gly Lys Ser

1 5

<210>69

<211>1035

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT3 coding sequence

<221> CDS

<222>(1)...(1035)

<400>69

atg cag gcg tac atg gcg gtc gcc gcc gct ccg gcg cca ccg gcc tcg 48

Met Gln Ala Tyr Met Ala Val Ala Ala Ala Pro Ala Pro Pro Ala Ser

1 5 10 15

ctg acg ctg ctg ccg cgc acc acc acc gtc atc agg gac agg gag cgc 96

Leu Thr Leu Leu Pro Arg Thr Thr Thr Val Ile Arg Asp Arg Glu Arg

20 25 30

ttc gac gcg gcc gtc ccg gtg gcg ccg ctc gtg ctg agg cat ggc gcc 144

Phe Asp Ala Ala Val Pro Val Ala Pro Leu Val Leu Arg His Gly Ala

35 40 45

ggc gtc aag cac aag gcc gtc gtg gtg atg ggc gcc acg ggc acc ggg 192

Gly Val Lys His Lys Ala Val Val Val Met Gly Ala Thr Gly Thr Gly

50 55 60

aag tca cgc ctc gcc gtc gac ctc gcc ctg cgg ttc ggc ggc gag gtc 240

Lys Ser Arg Leu Ala Val Asp Leu Ala Leu Arg Phe Gly Gly Glu Val

65 70 75 80

atc aac tcc gac aag atg cag ata cat tcc ggg ttg gat gtg gtg acg 288

Ile Asn Ser Asp Lys Met Gln Ile His Ser Gly Leu Asp Val Val Thr

85 90 95

aac aag gtg acc gag gag gag tgc gcc ggc gtg ccg cac cac ctg atc 336

Asn Lys Val Thr Glu Glu Glu Cys Ala Gly Val Pro His His Leu Ile

100 105 110

ggc gtg gcg cgc ccc gac gac gag ttc acg gcc gcc gac ttc cgc cgc 384

Gly Val Ala Arg Pro Asp Asp Glu Phe Thr Ala Ala Asp Phe Arg Arg

115 120 125

gag gcg gcg cgc gcc gcg gcg ggg gcg gtt gag agg ggg agg ttg ccc 432

Glu Ala Ala Arg Ala Ala Ala Gly Ala Val Glu Arg Gly Arg Leu Pro

130 135 140

atc atc gcc ggg ggg tcc aac tcc tac gtc gag gag ctc gtc gaa ggc 480

Ile Ile Ala Gly Gly Ser Asn Ser Tyr Val Glu Glu Leu Val Glu Gly

145 150 155 160

gac ggc cgc gcg ttc cgg gag cgg tgc tcc gcg gtt tcg tcg ccc gcc 528

Asp Gly Arg Ala Phe Arg Glu Arg Cys Ser Ala Val Ser Ser Pro Ala

165 170 175

gcg tcg acg aga tgt gcc ggc gag gcc tcg tcc ggg agg tgg ccg cag 576

Ala Ser Thr Arg Cys Ala Gly Glu Ala Ser Ser Gly Arg Trp Pro Gln

180 185 190

cgt tcg acc cgc gcc gca ccg act act ccc agg ggc atc tgg cgc gcc 624

Arg Ser Thr Arg Ala Ala Pro Thr Thr Pro Arg Gly Ile Trp Arg Ala

195 200 205

atc ggc gtg ccg gag ctc gac gcg tac ctc cgc tcc cgc ggc gac ggc 672

Ile Gly Val Pro Glu Leu Asp Ala Tyr Leu Arg Ser Arg Gly Asp Gly

210 215 220

gcc gac gag gag gag cgg gcg cgc atg ctc gcc gcg gcc gtc gcg gag 720

Ala Asp Glu Glu Glu Arg Ala Arg Met Leu Ala Ala Ala Val Ala Glu

225 230 235 240

atc aag tcg aac aag ttc cgg ctc gcg tgc cgc cag cac cgc aag atc 768

Ile Lys Ser Asn Thr Phe Arg Leu Ala Cys Arg Gln His Arg Lys Ile

245 250 255

gag agg ctg gac cgc atg tgg cgc gcc cgc cgc gtc gac gcc acg gag 816

Glu Arg Leu Asp Arg Met Trp Arg Ala Arg Arg Val Asp Ala Thr Glu

260 265 270

gtg ttc agg agg cgc ggc cac gcc gcc gac gac gcg tgg cag cgg ctc 864

Val Phe Arg Arg Arg Gly His Ala Ala Asp Asp Ala Trp Gln Arg Leu

275 280 285

gtc gcc gcg ccg tgc atc gac gcc gtc cgg tca ttc ctc ttc gag gac 912

Val Ala Ala Pro Cys Ile Asp Ala Val Arg Ser Phe Leu Phe Glu Asp

290 295 300

caa gaa cgc agc agc atc gcc gcc ggc aaa cct ccc ctc ttc gcc gcc 960

Gln Glu Arg Ser Ser Ile Ala Ala Gly Lys Pro Pro Leu Phe Ala Ala

305 310 315 320

ggc aag gcc act tca ggc aac atc tcc gtc ttc gcc tcc gcg gcc gcc 1008

Gly Lys Ala Thr Ser Gly Asn Ile Ser Val Phe Ala Ser Ala Ala Ala

325 330 335

gcc atg gcg gcg gcc gct gca at tga 1035

Ala Met Ala Ala Ala Ala Ala Ile *

340

<210>70

<211>1284

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT7 coding sequence

<221> CDS

<222>(1)...(1284)

<400>70

atg gca gcg act ggt cga aac gcg gcc gca cga cgg aca cgc cgc tcg 48

Met Ala Ala Thr Gly Arg Asn Ala Ala Ala Arg Arg Thr Arg Arg Ser

1 5 10 15

atc ccc cgc gcg gct gcc gtg ctc ccc ctc tcg tct gga tca ccg gcg 96

Ile Pro Arg Ala Ala Ala Val Leu Pro Leu Ser Ser Gly Ser Pro Ala

20 25 30

gct gtg ctg agg cga ctg ggg ctc ggt gag tgt ttt ggg tgg gcc ggc 144

Ala Val Leu Arg Arg Leu Gly Leu Gly Glu Cys Phe Gly Trp Ala Gly

35 40 45

ttt atg agc agt ctc ggt ttg aag atc cgc acc gtc gtc cgc tca cct 192

Phe Met Ser Ser Leu Gly Leu Lys Ile Arg Thr Val Val Arg Ser Pro

50 55 60

atg gcg gcc gcg gcc gtc gct ggc gtc gga agg gat ggt agc ttc gcc 240

Met Ala Ala Ala Ala Val Ala Gly Val Gly Arg Asp Gly Ser Phe Ala

65 70 75 80

tcc cag aag cgg cca cgt cgg gtt agt gtg aga atg gag aga agc aga 288

Ser Gln Lys Arg Pro Arg Arg Val Ser Val Arg Met Glu Arg Ser Arg

85 90 95

gtc ggg gac ggt tgc tgc tgc tcc tgc tct ggc cgc ggc ggg gtg gcg 336

Val Gly Asp Gly Cys Cys Cys Ser Cys Ser Gly Arg Gly Gly Val Ala

100 105 110

tcc act acg gcg gtc cgg ccg tcc acg ggg atg gtg gtg atc gtc ggc 384

Ser Thr Thr Ala Val Arg Pro Ser Thr Gly Met Val Val Ile Val Gly

115 120 125

gcc acg ggc acg ggg aag acc aag ctt tcc atc gac gcg gcg cag gag 432

Ala Thr Gly Thr Gly Lys Thr Lys Leu Ser Ile Asp Ala Ala Gln Glu

130 135 140

ctc gcc ggc gag gtg gtg aac gct gac aag att cag ctg tac gac ggc 480

Leu Ala Gly Glu Val Val Asn Ala Asp Lys Ile Gln Leu Tyr Asp Gly

145 150 155 160

ctc gac gtc acc acg aac aag gtg tcg ctc gcc gac cgc cgg ggc gtc 528

Leu Asp Val Thr Thr Asn Lys Val Ser Leu Ala Asp Arg Arg Gly Val

165 170 175

ccg cac cac ctc ctc ggc gca atc cgc gcc gag gcc ggg gag ctg ccg 576

Pro His His Leu Leu Gly Ala Ile Arg Ala Glu Ala Gly Glu Leu Pro

180 185 190

ccg tcg tcg ttc cgc tcg ctc gcc gcc gcc gcc gcg gcc ggc atc gcg 624

Pro Ser Ser Phe Arg Ser Leu Ala Ala Ala Ala Ala Ala Gly Ile Ala

195 200 205

tcg cgc ggg cgc gtg ccg gtc gtg gcc ggc ggg tcc aac tcg ctc atc 672

Ser Arg Gly Arg Val Pro Val Val Ala Gly Gly Ser Asn Ser Leu Ile

210 215 220

cac gcg ctc ctc gct gac ccc atc gat gcc gcg ccg cgt gac cct ttc 720

His Ala Leu Leu Ala Asp Pro Ile Asp Ala Ala Pro Arg Asp Pro Phe

225 230 235 240

gcg gac gcc gat gtc ggg tac cgg ccg gcg ctc cgg ttc ccg tgc tgc 768

Ala Asp Ala Asp Val Gly Tyr Arg Pro Ala Leu Arg Phe Pro Cys Cys

245 250 255

ctc ctc tgg gtc gac gtc gac gac gat gtt ctc gac gaa tac ctc gac 816

Leu Leu Trp Val Asp Val Asp Asp Asp Val Leu Asp Glu Tyr Leu Asp

260 265 270

cgg cgc gtg gac gac atg gtc ggc gag ggg atg gtc gag gag ctc gag 864

Arg Arg Val Asp Asp Met Val Gly Glu Gly Met Val Glu Glu Leu Glu

275 280 285

gaa tac ttc gcg acg acg tcg gcc tcg gag cgg gcc tcg cac gcc ggg 912

Glu Tyr Phe Ala Thr Thr Ser Ala Ser Glu Arg Ala Ser His Ala Gly

290 295 300

ctg ggc aag gcc atc ggc gtg ccg gag ctc ggc gac tac ttc gcc ggg 960

Leu Gly Lys Ala Ile Gly Val Pro Glu Leu Gly Asp Tyr Phe Ala Gly

305 310 315 320

cgc aag agc ctc gac gcg gcg ata gac gag atc aag gcc aac acg cgg 1008

Arg Lys Ser Leu Asp Ala Ala Ile Asp Glu Ile Lys Ala Asn Thr Arg

325 330 335

gtc ctc gcg gcc cgc cag gtc ggc aag atc cga cgc atg gcc gac gtt 1056

Val Leu Ala Ala Arg Gln Val Gly Lys Ile Arg Arg Met Ala Asp Val

340 345 350

tgg ggc tgg ccc atc cgc cgc ctc gac gcc acg gcc acc atc cgg gcg 1104

Trp Gly Trp Pro Ile Arg Arg Leu Asp Ala Thr Ala Thr Ile Arg Ala

355 360 365

cgg ctc tcc ggc gcc ggc cgc gcc gcc gag gcc gcc gcg tgg gag cgc 1152

Arg Leu Ser Gly Ala Gly Arg Ala Ala Glu Ala Ala Ala Trp Glu Arg

370 375 380

gac gtg cgc ggg cca ggc ctc gcc gcg atg cgt cag ttc gtc ggc cgc 1200

Asp Val Arg Gly Pro Gly Leu Ala Ala Met Arg Gln Phe Val Gly Arg

385 390 395 400

gcc gac ttc aac gcc gca gcg gtc gac cag cta gcc gcg cgg agt cgg 1248

Ala Asp Phe Asn Ala Ala Ala Val Asp Gln Leu Ala Ala Arg Ser Arg

405 410 415

agg caa tgc ctt cgc ggt ggc atg gtg gcc ggc tga 1284

Arg Gln Cys Leu Arg Gly Gly Met Val Ala Gly *

420 425

<210>71

<211>1353

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT8 coding sequence

<221> CDS

<222>(1)...(1353)

<400>71

atg gcc cac ctc gcg gcc tct gcc gcc ccg ctt cca agc gct gac ccc 48

Met Ala His Leu Ala Ala Ser Ala Ala Pro Leu Pro Ser Ala Asp Pro

1 5 10 15

gac gcc ggc gag gag tcc tcc cac tct ccg ccg ccg ccg gag aag ggg 96

Asp Ala Gly Glu Glu Ser Ser Ser His Ser Pro Pro Pro Pro Glu Lys Gly

20 25 30

ctg agg aag gtg gtg gtg gtg atg ggc gcg act ggc gcc ggc aag tcg 144

Leu Arg Lys Val Val Val Val Met Gly Ala Thr Gly Ala Gly Lys Ser

35 40 45

cgg ctg gcc gtc gac ctc gcg agc cac ttc gcc ggc gtc gag gtg gtc 192

Arg Leu Ala Val Asp Leu Ala Ser His Phe Ala Gly Val Glu Val Val

50 55 60

agc gcc gac tcc atg caa gtc tac ggt ggg ctc gat gtc ctc acc aac 240

Ser Ala Asp Ser Met Gln Val Tyr Gly Gly Leu Asp Val Leu Thr Asn

65 70 75 80

aag gtc ccc ctc cac gag cag aaa ggc gtt cct cac cat ctc ctg agc 288

Lys Val Pro Leu His Glu Gln Lys Gly Val Pro His His Leu Leu Ser

85 90 95

gtg att gat ccc tct gtg gag ttc act tgc cgc gat ttc cgc gac cat 336

Val Ile Asp Pro Ser Val Glu Phe Thr Cys Arg Asp Phe Arg Asp His

100 105 110

gct gtg ccg att ata gaa ggt ata ttg gat cgt ggc ggc ctc cct gtt 384

Ala Val Pro Ile Ile Glu Gly Ile Leu Asp Arg Gly Gly Leu Pro Val

115 120 125

att gtt ggt ggt aca aac ttc tac atc cag gct ctt gtt agc cca ttc 432

Ile Val Gly Gly Thr Asn Phe Tyr Ile Gln Ala Leu Val Ser Pro Phe

130 135 140

ctc ttt gat gat atg gca cag gat att gag ggt ctt act tta aat gac 480

Leu Phe Asp Asp Met Ala Gln Asp Ile Glu Gly Leu Thr Leu Asn Asp

145 150 155 160

cac cta gat gag ata ggg ctt gat aat gat gat gaa gcc ggt ctg tat 528

His Leu Asp Glu Ile Gly Leu Asp Asn Asp Asp Glu Ala Gly Leu Tyr

165 170 175

gaa cat ttg aag aag att gat cct gtt gct gca caa agg ata cac ccg 576

Glu His Leu Lys Lys Ile Asp Pro Val Ala Ala Gln Arg Ile His Pro

180 185 190

aac aac cat cga aaa ata aaa cgc tac ctt gag ttg tat gaa tcc aca 624

Asn Asn His Arg Lys Ile Lys Arg Tyr Leu Glu Leu Tyr Glu Ser Thr

195 200 205

ggt gcc cta cct agt gat ctt ttc caa ggg caa gcc aca gag gac aga 672

Gly Ala Leu Pro Ser Asp Leu Phe Gln Gly Gln Ala Thr Glu Asp Arg

210 215 220

agt ggg gtc gac cta gta act cca gat ttg act gtt gtt tct tgt gat 720

Ser Gly Val Asp Leu Val Thr Pro Asp Leu Thr Val Val Ser Cys Asp

225 230 235 240

gct gat ctt cat gtt ctg gat cgt tat gtc aat gaa agg gtc gac tgc 768

Ala Asp Leu His Val Leu Asp Arg Tyr Val Asn Glu Arg Val Asp Cys

245 250 255

atg att gat gat ggc ctg cta gat gaa gtg tgt aac ata tat gat cga 816

Met Ile Asp Asp Gly Leu Leu Asp Glu Val Cys Asn Ile Tyr Asp Arg

260 265 270

gag gcc act tat acc caa ggg ctg cgg cag gcc att ggt gtt cgt gaa 864

Glu Ala Thr Tyr Thr Gln Gly Leu Arg Gln Ala Ile Gly Val Arg Glu

275 280 285

ttt gat gag ttt ttc aga ttt tat ttt gca agg aag gaa acc ggt ctc 912

Phe Asp Glu Phe Phe Arg Phe Tyr Phe Ala Arg Lys Glu Thr Gly Leu

290 295 300

cat gat gat aac ctg aag ggc tta ttg gat gaa gca gtc tca caa cta 960

His Asp Asp Asn Leu Lys Gly Leu Leu Asp Glu Ala Val Ser Gln Leu

305 310 315 320

aaa gca aac act cgc aga ctt gtt cga cgt caa aga cga agg ctg cat 1008

Lys Ala Asn Thr Arg Arg Leu Val Arg Arg Gln Arg Arg Arg Leu His

325 330 335

cgg ttg aat aaa tat ttt gag tgg aac ttg cgt cat att gat gca aca 1056

Arg Leu Asn Lys Tyr Phe Glu Trp Asn Leu Arg His Ile Asp Ala Thr

340 345 350

gaa gct ttc tat ggt gcc act gct gac tca tgg aac atg aaa gtt gtg 1104

Glu Ala Phe Tyr Gly Ala Thr Ala Asp Ser Trp Asn Met Lys Val Val

355 360 365

aaa cct tgc gtg gat att gtt aga gat ttc ttg tct gat gat aca att 1152

Lys Pro Cys Val Asp Ile Val Arg Asp Phe Leu Ser Asp Asp Thr Ile

370 375 380

ttg gca agc aga gat ggt tct agt gta act gga agc cct agg atg tct 1200

Leu Ala Ser Arg Asp Gly Ser Ser Val Thr Gly Ser Pro Arg Met Ser

385 390 395 400

tca aga gag ttg tgg act caa tat gtt tgt gag gcc tgt gat aac cgg 1248

Ser Arg Glu Leu Trp Thr Gln Tyr Val Cys Glu Ala Cys Asp Asn Arg

405 410 415

gta ctt cgg gga acg cat gag tgg gag caa cac aag caa ggc cga tgc 1296

Val Leu Arg Gly Thr His Glu Trp Glu Gln His Lys Gln Gly Arg Cys

420 425 430

cac cgt aaa aga gta caa cgt ttg aaa cag aag gct agt aca gtg ata 1344

His Arg Lys Arg Val Gln Arg Leu Lys Gln Lys Ala Ser Thr Val Ile

435 440 445

tca tta tag 1353

Ser Leu *

450

<210>72

<211>1368

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT9 coding sequence

<221> CDS

<222>(1)...(1368)

<400>72

atg acc agc gtt gcc acc agg att gcc acg ctc gtg cgg gcc gcg gcg 48

Met Thr Ser Val Ala Thr Arg Ile Ala Thr Leu Val Arg Ala Ala Ala

1 5 10 15

gcg gcg agc cgg cca ttg cgg ctc cac cgc cgg ccc ggc ggc gag gat 96

Ala Ala Ser Arg Pro Leu Arg Leu His Arg Arg Pro Gly Gly Glu Asp

20 25 30

acg agg atg gtg gtg atc gtc ggc gcc acg ggc acc ggg aag acc aag 144

Thr Arg Met Val Val Ile Val Gly Ala Thr Gly Thr Gly Lys Thr Lys

35 40 45

ctg tcc atc gac gcc gcc aag gtg atc ggc ggc gag gtt gtc aac gcc 192

Leu Ser Ile Asp Ala Ala Lys Val Ile Gly Gly Glu Val Val Asn Ala

50 55 60

gac aag att cag ctc tat gac ggc ctc gac gtg acc acc aac aag gtg 240

Asp Lys Ile Gln Leu Tyr Asp Gly Leu Asp Val Thr Thr Asn Lys Val

65 70 75 80

agc ctc gcc gac cgc cgc ggc gtg ccg cac cac ctc ctc gga gcc atc 288

Ser Leu Ala Asp Arg Arg Gly Val Pro His His Leu Leu Gly Ala Ile

85 90 95

cgc ccc gag gcc ggc gag ctc ccg ccg tcg tcc ttc cgg tcc ctc gcc 336

Arg Pro Glu Ala Gly Glu Leu Pro Pro Pro Ser Ser Phe Arg Ser Leu Ala

100 105 110

gcc gcc acg gcc gcg tcg atc gcg gcg agg cgg ctc gtg ccg gtc atc 384

Ala Ala Thr Ala Ala Ser Ile Ala Ala Arg Arg Leu Val Pro Val Ile

115 120 125

gcc ggt ggg tcg aac tcc ctc atc cac gcc ctc ctc gcc gac cac ttc 432

Ala Gly Gly Ser Asn Ser Leu Ile His Ala Leu Leu Ala Asp His Phe

130 135 140

gac gcc tcc gct ggc gat ccc ttc tcc ccc gcc gcc gcc ttc cgc cac 480

Asp Ala Ser Ala Ala Gly Asp Pro Phe Ser Pro Ala Ala Ala Phe Arg His

145 150 155 160

tac cgc ccg gcg ctc cgg ttc ccg tgc tgc ctg ctc tgg gtc cac gtc 528

Tyr Arg Pro Ala Leu Arg Phe Pro Cys Cys Leu Leu Trp Val His Val

165 170 175

gat gag gcg ctc ctc gac gag tac ctc gac cgc cgc gtg gac gac atg 576

Asp Glu Ala Leu Leu Asp Glu Tyr Leu Asp Arg Arg Val Asp Asp Met

180 185 190

gtg gac gct ggc atg gtc gag gag ctc cgg gag tac ttc gcc acg aca 624

Val Asp Ala Gly Met Val Glu Glu Leu Arg Glu Tyr Phe Ala Thr Thr

195 200 205

acc gcc gcg gag cgc gcc gcg cac tcc ggg ctg ggc aag gcc atc ggc 672

Thr Ala Ala Glu Arg Ala Ala His Ser Gly Leu Gly Lys Ala Ile Gly

210 215 220

gtc ccc gag ctc ggc gac tac ttc gcc ggg cgc aag acc ttc tcc gag 720

Val Pro Glu Leu Gly Asp Tyr Phe Ala Gly Arg Lys Thr Phe Ser Glu

225 230 235 240

gcg atc gac gac atc aaa gcc aac acc cgc gtc ctc gcc gcc gcg cag 768

Ala Ile Asp Asp Ile Lys Ala Asn Thr Arg Val Leu Ala Ala Ala Gln

245 250 255

gtg tcc aag atc cgc cgc atg tcc gac gcc tgg ggc tgg ccc atc cac 816

Val Ser Lys Ile Arg Arg Met Ser Asp Ala Trp Gly Trp Pro Ile His

260 265 270

cgc ctc gac gcc tcc gac aca gtc cgc gcc agg ctc acg cgg gcg ggc 864

Arg Leu Asp Ala Ser Asp Thr Val Arg Ala Arg Leu Thr Arg Ala Gly

275 280 285

tcc gcc gcc gag tcc gcc tcc tgg gag cgc gac gtg cgc ggc cca ggc 912

Ser Ala Ala Glu Ser Ala Ser Trp Glu Arg Asp Val Arg Gly Pro Gly

290 295 300

ctc gcc acc atc cgc agc ttc ctc gcc gat cag tca ccg cca ccg cgc 960

Leu Ala Thr Ile Arg Ser Phe Leu Ala Asp Gln Ser Pro Pro Pro Arg

305 310 315 320

agc gag ggc acc aac gac tac ctg tac gcc atg gag acg gaa cca gag 1008

Ser Glu Gly Thr Asn Asp Tyr Leu Tyr Ala Met Glu Thr Glu Pro Glu

325 330 335

ccg ccg ccg ccg ccg acg ttg ccg ccg cgg ctg ctc cgg ttg ccg cgg 1056

Pro Pro Pro Pro Pro Thr Leu Pro Pro Arg Leu Leu Arg Leu Pro Arg

340 345 350

atg cag tac tgc gac atg gtg ggc aga att tgt gta ctc ttt ctt gga 1104

Met Gln Tyr Cys Asp Met Val Gly Arg Ile Cys Val Leu Phe Leu Gly

355 360 365

gag gtg gtg att cat cac atc gct ctc ctc cta acc gcg gca ctt ata 1152

Glu Val Val Ile His His Ile Ala Leu Leu Leu Thr Ala Ala Leu Ile

370 375 380

ctc cag tcc agt act ttt gcc tgt acc atc gct gca gtg tac gaa tgc 1200

Leu Gln Ser Ser Thr Phe Ala Cys Thr Ile Ala Ala Val Tyr Glu Cys

385 390 395 400

caa gtc tcc tgg ata atc gca agc ttt gcc tcc ggc cta ttt tgc agg 1248

Gln Val Ser Trp Ile Ile Ala Ser Phe Ala Ser Gly Leu Phe Cys Arg

405 410 415

att gct gta ctg gat gag aca gtc aag gag tta gat att tgg aag ctt 1296

Ile Ala Val Leu Asp Glu Thr Val Lys Glu Leu Asp Ile Trp Lys Leu

420 425 430

aag ata gcc aat gga ttt cta aaa att aca atg tgt ctg aaa gat tgg 1344

Lys Ile Ala Asn Gly Phe Leu Lys Ile Thr Met Cys Leu Lys Asp Trp

435 440 445

tca ggc tat ccc ctt gga gtg tga 1368

Ser Gly Tyr Pro Leu Gly Val *

450 455

<210>73

<211>1758

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT10 coding sequence

<221> CDS

<222>(1)...(1758)

<400>73

atg aag atc tac att caa gcc agg gaa gag aag tgt caa gga cat gtg 48

Met Lys Ile Tyr Ile Gln Ala Arg Glu Glu Lys Cys Gln Gly His Val

1 5 10 15

ttc aag tcc cgc cac ctt cga gcc aac aaa gag gca gca ctc cag gat 96

Phe Lys Ser Arg His Leu Arg Ala Asn Lys Glu Ala Ala Leu Gln Asp

20 25 30

gcg tcg cgt gag gca ttc atg cgt cta tgt aag atc tac agc atc gag 144

Ala Ser Arg Glu Ala Phe Met Arg Leu Cys Lys Ile Tyr Ser Ile Glu

35 40 45

gtt gcg agt act ccg ttc ttt cta cat cca ttc cgt gaa tgc ggt gac 192

Val Ala Ser Thr Pro Phe Phe Leu His Pro Phe Arg Glu Cys Gly Asp

50 55 60

cgc cgc tgc cat att cgg aaa ttt agg ggc ttt gag gag cac tct ccc 240

Arg Arg Cys His Ile Arg Lys Phe Arg Gly Phe Glu Glu His Ser Pro

65 70 75 80

atc cac ttc tcc atg tgg atg tgg gct gca gac gag gcc tat gag gag 288

Ile His Phe Ser Met Trp Met Trp Ala Ala Asp Glu Ala Tyr Glu Glu

85 90 95

gcc tta gag gaa tta gat atg ctt cgg tca aag att gcc ggc tgg gag 336

Ala Leu Glu Glu Leu Asp Met Leu Arg Ser Lys Ile Ala Gly Trp Glu

100 105 110

gag cgg tac aac cac ctt gct aaa gaa cac acc act cgt gga caa cta 384

Glu Arg Tyr Asn His Leu Ala Lys Glu His Thr Thr Arg Gly Gln Leu

115 120 125

ttg gaa gca atc aag ctt cgc ctc cag tgg tat ttt cga acc cca tct 432

Leu Glu Ala Ile Lys Leu Arg Leu Gln Trp Tyr Phe Arg Thr Pro Ser

130 135 140

caa gct caa atc caa cgg act ttg tca cca cca cca caa aga gtg aca 480

Gln Ala Gln Ile Gln Arg Thr Leu Ser Pro Pro Pro Gln Arg Val Thr

145 150 155 160

aga agt gat ggt gag gac tat agt caa atc aat gca cag cag gca tgt 528

Arg Ser Asp Gly Glu Asp Tyr Ser Gln Ile Asn Ala Gln Gln Ala Cys

165 170 175

ctg gaa agg tcc gaa gtt aaa ctt gat agg gca act tca caa gac tat 576

Leu Glu Arg Ser Glu Val Lys Leu Asp Arg Ala Thr Ser Gln Asp Tyr

180 185 190

ctg caa gga tac aag ccc cca tca gaa tcc ctc gac gct att gtt tgg 624

Leu Gln Gly Tyr Lys Pro Pro Ser Glu Ser Leu Asp Ala Ile Val Trp

195 200 205

cct ctt gtt gaa ggg aag cat gac aat aca agc agc ggt agg agg aat 672

Pro Leu Val Glu Gly Lys His Asp Asn Thr Ser Ser Gly Arg Arg Asn

210 215 220

gag aag gca tgg gaa atg gca aaa caa gtg cct aga gga agg att tat 720

Glu Lys Ala Trp Glu Met Ala Lys Gln Val Pro Arg Gly Arg Ile Tyr

225 230 235 240

gct ttg gtc tat aaa cat caa gac aat att gga ctg gtc ctt gaa aag 768

Ala Leu Val Tyr Lys His Gln Asp Asn Ile Gly Leu Val Leu Glu Lys

245 250 255

aga gtt gga cca gct gca cat cta gga gac cgc tat gta agt gac agg 816

Arg Val Gly Pro Ala Ala His Leu Gly Asp Arg Tyr Val Ser Asp Arg

260 265 270

gtc ata tca cat cta gga gac ctc tat gta ata cga acc ctt agc tac 864

Val Ile Ser His Leu Gly Asp Leu Tyr Val Ile Arg Thr Leu Ser Tyr

275 280 285

ttt cct ttc atg ctc aat ttt cac cct tct tgt gat tgc ttc ctc aat 912

Phe Pro Phe Met Leu Asn Phe His Pro Ser Cys Asp Cys Phe Leu Asn

290 295 300

atg ctg gga aac aag tta gta gtg att att ggt gcc acg gga act gga 960

Met Leu Gly Asn Lys Leu Val Val Ile Ile Gly Ala Thr Gly Thr Gly

305 310 315 320

aaa aca aga ctc tca att gag att gcc aag gcg att ggt ggg gaa gtg 1008

Lys Thr Arg Leu Ser Ile Glu Ile Ala Lys Ala Ile Gly Gly Glu Val

325 330 335

gta aat ggt gac aag atg caa att tat gat ggc ctg gat att acg aca 1056

Val Asn Gly Asp Lys Met Gln Ile Tyr Asp Gly Leu Asp Ile Thr Thr

340 345 350

aac aag gtt tct tta caa gat cga tgc ggc ata cct cat cac ctt att 1104

Asn Lys Val Ser Leu Gln Asp Arg Cys Gly Ile Pro His His Leu Ile

355 360 365

gcg tcc atc cct cac aac gca ggt gat ttt cct gtg tca ttt ttt cga 1152

Ala Ser Ile Pro His Asn Ala Gly Asp Phe Pro Val Ser Phe Phe Arg

370 375 380

tat gct gca aaa acc aca ata aac tgc att gcc aga cgt ggt cac aca 1200

Tyr Ala Ala Lys Thr Thr Ile Asn Cys Ile Ala Arg Arg Gly His Thr

385 390 395 400

ccg att gtg gtg ggt gga tct aac tca ctt atc cat ggt ctc ctt gtt 1248

Pro Ile Val Val Gly Gly Ser Asn Ser Leu Ile His Gly Leu Leu Val

405 410 415

gac aat ttt gat ttg tct att gtg gat cct ttt ggg caa ttg gag gtt 1296

Asp Asn Phe Asp Leu Ser Ile Val Asp Pro Phe Gly Gln Leu Glu Val

420 425 430

agc tat cag ccg acg cct caa tgg caa tgt tgt ttt cta tgg gtt cat 1344

Ser Tyr Gln Pro Thr Pro Gln Trp Gln Cys Cys Phe Leu Trp Val His

435 440 445

gtt aat gag gtg att ctt aat gag tat ttg aaa cgt cgt gtt gac ggc 1392

Val Asn Glu Val Ile Leu Asn Glu Tyr Leu Lys Arg Arg Val Asp Gly

450 455 460

atg gtt gat gct ggg tta gtt gag gaa att gaa gaa tat ttt gac aca 1440

Met Val Asp Ala Gly Leu Val Glu Glu Ile Glu Glu Tyr Phe Asp Thr

465 470 475 480

tta tca gtt aat gga cat gtt cca tat gtg gga tta ggg aag gcc att 1488

Leu Ser Val Asn Gly His Val Pro Tyr Val Gly Leu Gly Lys Ala Ile

485 490 495

ggt gtt cca gag cta agc gag tat ttt act gga cgg gtg agt tgt agt 1536

Gly Val Pro Glu Leu Ser Glu Tyr Phe Thr Gly Arg Val Ser Cys Ser

500 505 510

gat gct ctt tct atg atg aag acc aat aca cag att ctt gca cga tct 1584

Asp Ala Leu Ser Met Met Lys Thr Asn Thr Gln Ile Leu Ala Arg Ser

515 520 525

caa gtc aca aag att cat cgc atg gtt gat gtg tgg gga tgg cat gtt 1632

Gln Val Thr Lys Ile His Arg Met Val Asp Val Trp Gly Trp His Val

530 535 540

cat gcc ctt gat tgt act gaa act att cta gca cat ctt act gga tca 1680

His Ala Leu Asp Cys Thr Glu Thr Ile Leu Ala His Leu Thr Gly Ser

545 550 555 560

aat aag tat atg gag gat ttg gtg tgg aaa cgt gat gta agt gac tct 1728

Asn Lys Tyr Met Glu Asp Leu Val Trp Lys Arg Asp Val Ser Asp Ser

565 570 575

gga ctt gct gct ata caa gat ttt ctg tga 1758

Gly Leu Ala Ala Ile Gln Asp Phe Leu *

580 585

<210>74

<211>1773

<212>DNA

<213> Rice

<220>

<221>misc_feature

<222>(0)...(0)

<223> OsIPT11 coding sequence

<221> CDS

<222>(1)...(1773)

<400>74

atg gag aac tcc tca aag aaa acc caa gag ttc ttc cct aaa ggt ggg 48

Met Glu Asn Ser Ser Lys Lys Thr Gln Glu Phe Phe Pro Lys Gly Gly

1 5 10 15

aat gga ggt tat gct gag cag ctg gag ctc ttg ctg aag cag ctt cgt 96

Asn Gly Gly Tyr Ala Glu Gln Leu Glu Leu Leu Leu Lys Gln Leu Arg

20 25 30

ttt cct aac aag ccg atc cac cat gcg gag caa gtg atc aaa gga ttc 144

Phe Pro Asn Lys Pro Ile His His Ala Glu Gln Val Ile Lys Gly Phe

35 40 45

cgg aag gat tgg acg atg aag atc tac att caa gcc agg gaa gag aag 192

Arg Lys Asp Trp Thr Met Lys Ile Tyr Ile Gln Ala Arg Glu Glu Lys

50 55 60

tgt caa gga cat gtg ttc aag tcc cgc cac ctt cga gcc aac aaa gag 240

Cys Gln Gly His Val Phe Lys Ser Arg His Leu Arg Ala Asn Lys Glu

65 70 75 80

gca gca ctc cag gat gcg tcg cgt gag gca ttc atg cgt cta tgt aag 288

Ala Ala Leu Gln Asp Ala Ser Arg Glu Ala Phe Met Arg Leu Cys Lys

85 90 95

atc tac agc atc gag gtt gca agt act ccg ttc ttt cta cat cca ttc 336

Ile Tyr Ser Ile Glu Val Ala Ser Thr Pro Phe Phe Leu His Pro Phe

100 105 110

cgt gaa tgc ggt gac cgc cgc tgc cat att cgg aaa ttt agg ggc ttt 384

Arg Glu Cys Gly Asp Arg Arg Cys His Ile Arg Lys Phe Arg Gly Phe

115 120 125

gag gag cag tcg ccc atc cac ttc tcc atg tgg atg tgg gct gca gac 432

Glu Glu Gln Ser Pro Ile His Phe Ser Met Trp Met Trp Ala Ala Asp

130 135 140

gag gcc tat gag gag gcc tta gag gaa tta gat atg ctt cgg tca aag 480

Glu Ala Tyr Glu Glu Ala Leu Glu Glu Leu Asp Met Leu Arg Ser Lys

145 150 155 160

atc gcc ggc tgg gag gag cgg tac aac cac ctt gct aaa gaa cac acc 528

Ile Ala Gly Trp Glu Glu Arg Tyr Asn His Leu Ala Lys Glu His Thr

165 170 175

act cgt gga caa cta ttg gaa gca atc aag ctt cgc ctc cag tgg tat 576

Thr Arg Gly Gln Leu Leu Glu Ala Ile Lys Leu Arg Leu Gln Trp Tyr

180 185 190

ttt cga acc cca tct caa gct cat atc caa cgg act ttg cca cca cca 624

Phe Arg Thr Pro Ser Gln Ala His Ile Gln Arg Thr Leu Pro Pro Pro

195 200 205

cca caa aga gtg aca aga agt gat ggt gag gac tat agt caa atc aat 672

Pro Gln Arg Val Thr Arg Ser Asp Gly Glu Asp Tyr Ser Gln Ile Asn

210 215 220

gca cat cag gca tgt ctg gaa agg tcc gaa gtt aaa ctt gat agg gca 720

Ala His Gln Ala Cys Leu Glu Arg Ser Glu Val Lys Leu Asp Arg Ala

225 230 235 240

act tca caa gac tat ctg caa gga tac aag ccc cca tca gaa tcc ctc 768

Thr Ser Gln Asp Tyr Leu Gln Gly Tyr Lys Pro Pro Ser Glu Ser Leu

245 250 255

gac gct att gtt tgg cct ctt gtt gaa ggg aag cat gac aat aca agc 816

Asp Ala Ile Val Trp Pro Leu Val Glu Gly Lys His Asp Asn Thr Ser

260 265 270

agt ggt agg agg aat gag aag gca tgg gaa atg gca aaa caa gta ata 864

Ser Gly Arg Arg Asn Glu Lys Ala Trp Glu Met Ala Lys Gln Val Ile

275 280 285

cga acc ctt agc tac ttt cct ttc atg ctc aat ttt cac cct tct tgt 912

Arg Thr Leu Ser Tyr Phe Pro Phe Met Leu Asn Phe His Pro Ser Cys

290 295 300

gat tgc ttc ctc aat atg ctg gga aac aag tta gta gtg att att ggt 960

Asp Cys Phe Leu Asn Met Leu Gly Asn Lys Leu Val Val Ile Ile Gly

305 310 315 320

gcc acg gga act gga aaa aca aga ctc tca att gag ata gcc aag gcg 1008

Ala Thr Gly Thr Gly Lys Thr Arg Leu Ser Ile Glu Ile Ala Lys Ala

325 330 335

att ggt ggg gaa gtg gta aat gct gac aag atg caa att tac gat ggc 1056

Ile Gly Gly Glu Val Val Asn Ala Asp Lys Met Gln Ile Tyr Asp Gly

340 345 350

ctg gat att acg aca aac aag gtt tct tta caa gat cga tgc ggcata 1104

Leu Asp Ile Thr Thr Asn Lys Val Ser Leu Gln Asp Arg Cys Gly Ile

355 360 365

cct cat cac ctt att gcg tcc atc cct cgc aac gca ggt gat ttt cct 1152

Pro His His Leu Ile Ala Ser Ile Pro Arg Asn Ala Gly Asp Phe Pro

370 375 380

gtg tca ttt ttt cga tct gct gca aaa acc aca ata aac tgc att gcc 1200

Val Ser Phe Phe Arg Ser Ala Ala Lys Thr Thr Ile Asn Cys Ile Ala

385 390 395 400

aga cgt ggt cac aca ccg att gtg gtg ggt gga tct aac tca ctt atc 1248

Arg Arg Gly His Thr Pro Ile Val Val Gly Gly Ser Asn Ser Leu Ile

405 410 415

cat ggt ctc ctt gtt gac aat ttt gat tcg tct att gtg gat cct ttt 1296

His Gly Leu Leu Val Asp Asn Phe Asp Ser Ser Ile Val Asp Pro Phe

420 425 430

ggg caa ttg gag gtt agc tat cgg ccg acg cct cga tcg caa tgt tgt 1344

Gly Gln Leu Glu Val Ser Tyr Arg Pro Thr Pro Arg Ser Gln Cys Cys

435 440 445

ttt cta tgg gtt cat gtt aat gag gtg att ctt aat gag tat ttg aaa 1392

Phe Leu Trp Val His Val Asn Glu Val Ile Leu Asn Glu Tyr Leu Lys

450 455 460

cgt cgt gtt gac aac atg gtt gat gct ggg tta gtt gag gaa att gaa 1440

Arg Arg Val Asp Asn Met Val Asp Ala Gly Leu Val Glu Glu Ile Glu

465 470 475 480

gaa tat ttt gac aca tta tca gtt aat gga cat gtt cca tat gtg gga 1488

Glu Tyr Phe Asp Thr Leu Ser Val Asn Gly His Val Pro Tyr Val Gly

485 490 495

tta ggg aag gcc att ggt gtt cca gag cta agc gag tat ttt act gga 1536

Leu Gly Lys Ala Ile Gly Val Pro Glu Leu Ser Glu Tyr Phe Thr Gly

500 505 510

cgg gtg agt tgt agt gat gct ctt tct atg atg aag acc aat aca cag 1584

Arg Val Ser Cys Ser Asp Ala Leu Ser Met Met Lys Thr Asn Thr Gln

515 520 525

att ctt gca cga tct caa gtc aca aag att cat cgc atg gtt gat gtg 1632

Ile Leu Ala Arg Ser Gln Val Thr Lys Ile His Arg Met Val Asp Val

530 535 540

tgg gga tgg cat gtt cat gcc ctt gat tgt act gaa act att cta gca 1680

Trp Gly Trp His Val His Ala Leu Asp Cys Thr Glu Thr Ile Leu Ala

545 550 555 560

cat ctt act gga tca aat aag tat atg gag gat ttg gtg tgg aaa cgt 1728

His Leu Thr Gly Ser Asn Lys Tyr Met Glu Asp Leu Val Trp Lys Arg

565 570 575

gat gta agt gac cct gga ctt gct gct ata caa gat ttt ctg tga 1773

Asp Val Ser Asp Pro Gly Leu Ala Ala Ile Gln Asp Phe Leu *

580 585 590

<210>75

<211>3280

<212>DNA

<213> corn

<220>

<221> promoter

<222>(1)...(3280)

<223> ZmlPT2 promoter (from Mo17)

<400>75

tagtcagaga ggagccaaca acaatgccct ttgaggtggt taaaacctag agggtgaaga 60

agggcttctt tggccatcaa tgaaggatta taaaatcatg gaaccacttg ttactcaatc 120

tctcactatt tgtctattcg atgtgtgatg aagcgttgat gatacattgt gtgttggcac 180

atgggcttgt gttctactca ttacgtaggc catgtgatag gtgagtagtg gctaactcga 240

caagtggtga tcaagtgagg tagggttaca tagtttttat atatatgata ttatttgggc 300

atatattata cctatattat tgggcttatt ttggtcagta tttgaaatgc attctataat 360

gttcgaaggc cctaacgggt aaccaagaag ggtacatagg aaacaggttg attgtaatgt 420

attcgatatc cttctaaaat ttggccataa agccctttat ggggagtgaa aacaacccat 480

caatgtctcg taacggagaa atccctatta tttataaggg attgggcccc atttccatgc 540

ctacggaatg cttatattac aagtgcactc tagaaatgta acttgcacat ggatgacatg 600

attaggattg gtggcatgtg atacaattcc ttttttatt ccacattttt atttgacttt 660

ggtttttagt attttttgtt tgtcctggac acccggtcac tggagtccac ttccttgtca 720

atgaacctta actacccacc accaaaaaat ccctctttct actttcatta tattggtata 780

attgctacag ctaccttgtt agttgcaaaa gactagtccc attgccttac tagtgaccct 840

aatggagggc tacatatcct tggtagatgt ggaggtacca atggttccat cacatccatt 900

agattaggag gacaccatga tagacactag tctcaatatt aaacatagtt cttgttttta 960

ttttaaaatc gaaagcatta ttttgtttta aattctttta gtcgaaataa acttttaaaa 1020

cttcctagtt caatgaagtt tttctaaact tgaaactatg tgtattgttt gtacttgaaa 1080

cttttgactg gagagtttat acttgttggc atttatatga ttgctttatg cttaggacaa 1140

tatacctatt gggcttattt aacttggtca gcgcatgaaa ctctttgtat aaattccaaa 1200

ggcccttgat gggcactcgc tttttattcc ttcgggtagc aaaaacaagg caacacaagt 1260

aagaatgatt aaagcttctc cgcttaatag cttctccttc tgaaatctac cgaggagaga 1320

aggtaaagga gaagctttga cagcttctcc cctcgataac ttctccttct aaaatctatc 1380

aaggggagaa ggtaaagaag aagctttgac atctctttaa ttactaatca aaggacaaaa 1440

caaaagagat tcttattcaa tacattcccg tctagaatca gctttcattc ttgcaacgca 1500

acaacaatta caaatatacc ttctacctcg gtaataggag aaggtatctt tggaggcaga 1560

gcagattagt gttccaagtt cctgctccct ctcaaatgca tggtattgtt gctctattta 1620

tagccacggg gtacagcttt gtatgaaatt acaaacatac ccacaaactt atacaattgg 1680

actaatgaat acataagggg taatgcagtc atttttgttc acttgcctcg ccaatcgggt 1740

ctcttgggtt tctcaccttc ttctcttctt tgatcttcgt tatgtgttgg tcgaagcttc 1800

cttcggcaca taccttcgtg gttggtgctt tgaagcttct ccttcctagt ttttaagatt 1860

ttccaaagga gcttctcctt cctagttttg aagcttctcc gaaggatctt ctcctttcta 1920

gtttttaagc ttctccaaag tagcttctcc tttgcatagt acttgaaatg tattcaatgt 1980

taaagggttt ttgaggatct tcggtgatag aggccctcca atagccgtca acatgggcca 2040

gtaattggga agggtacaca agaaatggta gcctaaccccc atcatatata atatagggat 2100

aaattttggc catatagccc ttagtgggga gtgtaacaaa cctatcaagg cccctattcg 2160

gaggaaatcc tctacagaga ttggggcaca ttgtcatacc tattgagatg tttattaaac 2220

acatgcactc ataatgttta tttaaatgta acttgtggat cgatgacgtg atcaagacct 2280

atttttttca cagacctatt tctcctattt tcatccacat tcgtgtaatc tcatttgtct 2340

ctcgtctcta gtcttttctt ttaagttgga aacattattt gtttttaaat cctttctagt 2400

caaagtttta gacaagggaa catatcaggt gcaaccatac ccccattagtg taaccgtgca 2460

accaagacac aagaatggtg gtgggaggac cattttaaaa aaattctctt ccacctccat 2520

ccttgtcttg gttgcacggt agcaccaaca gatatggttg catctcatat atatgttccc 2580

tttaaacaaa tggttccatt taaaactttc tagttgaatg aagttttttt ctaaactcaa 2640

ctctttttgt attatttgta agtgaaaatt ttgactttgc gtgtttagac ttcaaactta 2700

attatttcct gctgtgctag aggacaacta gtaccaaaac ccaccaagtt cagtcaggta 2760

gaaatttact caagattatg atatggtcgt ttctttgatt ggagtcaatc gatggagtcc 2820

atgaacccaa acatttccat cggcagtatt gaacgaagat ggcagagtaa agtttggtaa 2880

tctttagtgg ttacaattat agaaatctcg aaacattttt tgaagcaggt aataatgcat 2940

gagctctaaa gaaggaaaaa tataatatct gttaacacat agaatcgagt gctccaatag 3000

atttagatat taacatatat gcattggata tatggaaaaa aaggatactc atgatatgag 3060

cacattaaat ttcctccaag caaacctagt ataaaaaggg aggaatgggt agatgataga 3120

gcagctcgtt ccttaaacca aatacaccac aaatttcttc aaaacaaaca aacacgatac 3180

atactggtct ctgtgcacaa aaaaggcacg gactgcttct ttttctattt ttttgttgtg 3240

tgcacagaat cgagcggcta cgataatcaa gatcaagaca 3280

<210>76

<211>969

<212>DNA

<213> corn

<220>

<221>misc_feature

<222>(0)...(0)

Coding sequence for variant of <223> ZmlPT2 (from Mo17)

<221> CDS

<222>(1)...(969)

<400>76

atg gag cac ggt gcc gtc gcc ggg aag ccc aag gtg gtg ttc gtg ctc 48

Met Glu His Gly Ala Val Ala Gly Lys Pro Lys Val Val Phe Val Leu

1 5 10 15

ggc gcg aca gcg aca ggg aag tcg aag ctc gcc atc gcc ctc gcc gag 96

Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu Ala Ile Ala Leu Ala Glu

20 25 30

cgc ttc aac ggt gag gtt atc aac gct gac aaa atc cag gtc cac gat 144

Arg Phe Asn Gly Glu Val Ile Asn Ala Asp Lys Ile Gln Val His Asp

35 40 45

ggc gtg ccc atc atc acg aac aag gtc aca gag gaa gag cag ggc ggg 192

Gly Val Pro Ile Ile Thr Asn Lys Val Thr Glu Glu Glu Gln Gly Gly

50 55 60

gtg ccc cac cac ctg ctc agc gtc cgc cac ccg gac gcc gac ttc act 240

Val Pro His His Leu Leu Ser Val Arg His Pro Asp Ala Asp Phe Thr

65 70 75 80

gcg gag gag ttc cga cgt gag gcg gcc agc gcc gtg gcc cgc gtg ctc 288

Ala Glu Glu Phe Arg Arg Glu Ala Ala Ser Ala Val Ala Arg Val Leu

85 90 95

tcg gcg ggc cgc ctc ccc gtc gtg gca ggc ggg tcc aac acc tac atc 336

Ser Ala Gly Arg Leu Pro Val Val Ala Gly Gly Ser Asn Thr Tyr Ile

100 105 110

gag gca ctg gtg gaa ggc gac ggt gcc gcc ttc cgc ttg gcg cac gac 384

Glu Ala Leu Val Glu Gly Asp Gly Ala Ala Phe Arg Leu Ala His Asp

115 120 125

ctc ctc ttc gtc tgg gtg gac gcg gag cgg gag ctg ttg gag tgg tac 432

Leu Leu Phe Val Trp Val Asp Ala Glu Arg Glu Leu Leu Glu Trp Tyr

130 135 140

gcc gcg ctg cgc gtg gac gag atg gtg gcc cgc ggg ctg gtg agc gag 480

Ala Ala Leu Arg Val Asp Glu Met Val Ala Arg Gly Leu Val Ser Glu

145 150 155 160

gct cgc gcg gcg ttt ggc ggc gcc gga gtt gac tac aac cat ggc gtg 528

Ala Arg Ala Ala Phe Gly Gly Ala Gly Val Asp Tyr Asn His Gly Val

165 170 175

cgc cgc gcc atc ggc ctg ccg gag atg cac gcc tac ctg gtg gcg gag 576

Arg Arg Ala Ile Gly Leu Pro Glu Met His Ala Tyr Leu Val Ala Glu

180 185 190

cac gag ggc gtc gcc ggg gag gcc gag ctc gcg gcc atg ctg gaa cgc 624

His Glu Gly Val Ala Gly Glu Ala Glu Leu Ala Ala Met Leu Glu Arg

195 200 205

gcg gtg cgc gag atc aag gac aac acc ttc cgc ctc gcg cgc acg cag 672

Ala Val Arg Glu Ile Lys Asp Asn Thr Phe Arg Leu Ala Arg Thr Gln

210 215 220

gcg gag aag atc cgg cgc ctc agc acg ctt gac ggc tgg gac gtc cgc 720

Ala Glu Lys Ile Arg Arg Leu Ser Thr Leu Asp Gly Trp Asp Val Arg

225 230 235 240

cgc atc gac gtg acc ccc gtg ttc gcg cgc aag gcc gat ggc act gag 768

Arg Ile Asp Val Thr Pro Val Phe Ala Arg Lys Ala Asp Gly Thr Glu

245 250 255

tgc cac gag ctg act tgg aag aag cag gtg tgg gag ccg tgc gag gag 816

Cys His Glu Leu Thr Trp Lys Lys Gln Val Trp Glu Pro Cys Glu Glu

260 265 270

atg gtg agg gct ttc ctc gag ccg tcc ctg act gcc gtt cca ggt gtt 864

Met Val Arg Ala Phe Leu Glu Pro Ser Leu Thr Ala Val Pro Gly Val

275 280 285

gca gta act gaa gaa ggg aac gcc ggc gtc gtc gct act gct gca ccc 912

Ala Val Thr Glu Glu Gly Asn Ala Gly Val Val Ala Thr Ala Ala Pro

290 295 300

gct ggt gat gtc gtc gtc cca act ggc gat gtc gtc acc gcc gtg gct 960

Ala Gly Asp Val Val Val Pro Thr Gly Asp Val Val Thr Ala Val Ala

305 310 315 320

gat gca taa 969

Asp Ala *

<210>77

<211>322

<212>PRT

<213> corn

<400>77

Met Glu His Gly Ala Val Ala Gly Lys Pro Lys Val Val Phe Val Leu

1 5 10 15

Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu Ala Ile Ala Leu Ala Glu

20 25 30

Arg Phe Asn Gly Glu Val Ile Asn Ala Asp Lys Ile Gln Val His Asp

35 40 45

Gly Val Pro Ile Ile Thr Asn Lys Val Thr Glu Glu Glu Gln Gly Gly

50 55 60

Val Pro His His Leu Leu Ser Val Arg His Pro Asp Ala Asp Phe Thr

65 70 75 80

Ala Glu Glu Phe Arg Arg Glu Ala Ala Ser Ala Val Ala Arg Val Leu

85 90 95

Ser Ala Gly Arg Leu Pro Val Val Ala Gly Gly Ser Asn Thr Tyr Ile

100 105 110

Glu Ala Leu Val Glu Gly Asp Gly Ala Ala Phe Arg Leu Ala His Asp

115 120 125

Leu Leu Phe Val Trp Val Asp Ala Glu Arg Glu Leu Leu Glu Trp Tyr

130 135 140

Ala Ala Leu Arg Val Asp Glu Met Val Ala Arg Gly Leu Val Ser Glu

145 150 155 160

Ala Arg Ala Ala Phe Gly Gly Ala Gly Val Asp Tyr Asn His Gly Val

165 170 175

Arg Arg Ala Ile Gly Leu Pro Glu Met His Ala Tyr Leu Val Ala Glu

180 185 190

His Glu Gly Val Ala Gly Glu Ala Glu Leu Ala Ala Met Leu Glu Arg

195 200 205

Ala Val Arg Glu Ile Lys Asp Asn Thr Phe Arg Leu Ala Arg Thr Gln

210 215 220

Ala Glu Lys Ile Arg Arg Leu Ser Thr Leu Asp Gly Trp Asp Val Arg

225 230 235 240

Arg Ile Asp Val Thr Pro Val Phe Ala Arg Lys Ala Asp Gly Thr Glu

245 250 255

Cys His Glu Leu Thr Trp Lys Lys Gln Val Trp Glu Pro Cys Glu Glu

260 265 270

Met Val Arg Ala Phe Leu Glu Pro Ser Leu Thr Ala Val Pro Gly Val

275 280 285

Ala Val Thr Glu Glu Gly Asn Ala Gly Val Val Ala Thr Ala Ala Pro

290 295 300

Ala Gly Asp Val Val Val Pro Thr Gly Asp Val Val Thr Ala Val Ala

305 310 315 320

Asp Ala

<210>78

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>78

atcatcaaga caatggagca cggtg 25

<210>79

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>79

cgtccgctag ctacttatgc atcag 25

<210>80

<211>53

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>80

ggggacaagt ttgtacaaaa aagcaggctc aatggagcac ggtgccgtcg ccg 53

<210>81

<211>52

<212>DNA

<213> Artificial sequence

<220>

<223> Primer

<400>81

ggggaccact ttgtacaaga aagctgggtc ttatgcatca gccacggcgg tg 52

<210>82

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>ZmIPT2-F primer

<400>82

tgttgtgtgc acagaatcga gcgg 24

<210>83

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223>ZmIPT2-R primer

<400>83

cgtccgctag ctacttatgc atcag 25

<210>84

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> Mu TIR primer

<400>84

agagaagcca acgccawcgc ctcyatttcg tc 32

<210>85

<211>1495

<212>DNA

<213> corn

<220>

<221> CDS

<222>(83)...(1048)

<223> ZmIPT2 ORF

<221>polyA_signal

<222>(1411)...(1416)

<221>misc_feature

<222>(1214)...(1230)

<223> Lynx mark

<221>misc_feature

<222>(624)...(632)

<223> transposon

<400>85

aaaaggcacg gactgcttct ttttctattt tttgttgtgt gcacagaatc gagcggctac 60

aataatcaag atcatcaaga ca atg gag cac ggt gcc gtc gcc ggg aag ccc 112

                                                                                        , 

1 5 10

aag gtg gtg ttc gtg ctc ggc gcc aca gcg aca ggg aag tcg aag ctc 160

Lys Val Val Phe Val Leu Gly Ala Thr Ala Thr Gly Lys Ser Lys Leu

15 20 25

gcc atc gcc ctc gcc gag cgc ttc aac ggt gag gtt atc aac gct gac 208

Ala Ile Ala Leu Ala Glu Arg Phe Asn Gly Glu Val Ile Asn Ala Asp

30 35 40

aaa atc cag gtc cac gat ggc gtg ccc atc atc acg aac aag gtc aca 256

Lys Ile Gln Val His Asp Gly Val Pro Ile Ile Thr Asn Lys Val Thr

45 50 55

gag gaa gag cag ggc ggg gtg ccc cac cac ctg ctc agc gtc cgc cac 304

Glu Glu Glu Gln Gly Gly Val Pro His His Leu Leu Ser Val Arg His

60 65 70

ccg gac gcc gac ttc act gcg gag gag ttc cga cgt gag gcg gcc agc 352

Pro Asp Ala Asp Phe Thr Ala Glu Glu Phe Arg Arg Glu Ala Ala Ser

75 80 85 90

gcc gtg gcc cgc gtg ctc tcg gcg ggc cgc ctc ccc gtc gtg gca ggc 400

Ala Val Ala Arg Val Leu Ser Ala Gly Arg Leu Pro Val Val Ala Gly

95 100 105

ggg tcc aac acc tac atc gag gca ctg gtg gaa ggc gac ggc gcc gcc 448

Gly Ser Asn Thr Tyr Ile Glu Ala Leu Val Glu Gly Asp Gly Ala Ala

110 115 120

ttc cgc gcg gcg cac gac ctc ctc ttc gtc tgg gtg gac gcg gag cag 496

Phe Arg Ala Ala His Asp Leu Leu Phe Val Trp Val Asp Ala Glu Gln

125 130 135

gag ctg ctg gag tgg tac gcc gcg ctg cgc gtg gac gag atg gtg gcc 544

Glu Leu Leu Glu Trp Tyr Ala Ala Leu Arg Val Asp Glu Met Val Ala

140 145 150

cgc ggg ctg gtg agc gag gct cgc gcg gcg ttc ggc ggc gcc ggg gtt 592

Arg Gly Leu Val Ser Glu Ala Arg Ala Ala Phe Gly Gly Ala Gly Val

155 160 165 170

gac tac aac cat ggc gtg cgc cgc gcc atc ggc ctg ccg gag atg cac 640

Asp Tyr Asn His Gly Val Arg Arg Ala Ile Gly Leu Pro Glu Met His

175 180 185

gcc tac ctg gtg gcg gag cgc gag ggc gtc gct ggg gag gcc gag ctc 688

Ala Tyr Leu Val Ala Glu Arg Glu Gly Val Ala Gly Glu Ala Glu Leu

190 195 200

gcg gcc atg ctg gaa cgc gcg gtg cgc gag atc aag gac aac acc ttc 736

Ala Ala Met Leu Glu Arg Ala Val Arg Glu Ile Lys Asp Asn Thr Phe

205 210 215

cgc ctc gcg cgc acg cag gcg gag aag atc cgg cgc ctc agc acg ctc 784

Arg Leu Ala Arg Thr Gln Ala Glu Lys Ile Arg Arg Leu Ser Thr Leu

220 225 230

gac ggc tgg gac gtc cgc cgc atc gac gtg acc ccc gtg ttc gcg cgc 832

Asp Gly Trp Asp Val Arg Arg Ile Asp Val Thr Pro Val Phe Ala Arg

235 240 245 250

aag gcc gat ggc act gag tgc cac gag ctg act tgg aag aag cag gtg 880

Lys Ala Asp Gly Thr Glu Cys His Glu Leu Thr Trp Lys Lys Gln Val

255 260 265

tgg gag ccg tgc gag gag atg gtg agg gct ttc ctc gag ccg tcc ctg 928

Trp Glu Pro Cys Glu Glu Met Val Arg Ala Phe Leu Glu Pro Ser Leu

270 275 280

act gcc gtt cca ggt gtt gca gta act gaa gaa ggg aac gcc ggc gtc 976

Thr Ala Val Pro Gly Val Ala Val Thr Glu Glu Gly Asn Ala Gly Val

285 290 295

gtc gct act gct gca ccc gct ggt gat gtc gtc gtc cca act ggc gat 1024

Val Ala Thr Ala Ala Pro Ala Gly Asp Val Val Val Pro Thr Gly Asp

300 305 310

gtc gtc acc gcc gtg gct gat gca taagtagcta gcggacgtag cgcatgcatg 1078

Val Val Thr Ala Val Ala Asp Ala

315 320

caatgcatgc aggctggctg gctggcttaa ttagtgcctc cgacttgctt taaactcatg 1138

tagctgcgtc catgggagag ggtgagatac aagtttatgc gacttatatt tctttctaaa 1198

tttaaatgga tctcggatcc gtagtatctg gtttaatata attataatat ttccttcgaa 1258

ttattatata tatatgctca cactcagtta gggatatata ctccctccat tcactctatg 1318

tatttggatt catatgcaaa agtattttaa aattatacta cctccattct cgaatatttg 1378

ttacccgctt gtttattttc taaaacatga taaataaaaa aacggagaga atagtatttt 1438

attatttgtt gatgatatat tttgtaagat atgaacggtg aaagttttac cataaag 1495

<210>86

<211>71

<212>DNA

<213> Artificial sequence

<220>

<223>IPT2 TIR L

<400>86

gagataattg ccattataga agaagagaga aggggattcg acgaaataga ggcgatggcg 60

ttggcttctc t 71

<210>87

<211>67

<212>DNA

<213> Artificial sequence

<220>

<223>IPT2 TIR R

<400>87

aagccaacgc caacgcctct atttcgtcga atccccttct ctcttcttct ataatggcaa 60

ttatctc 67

Claims (32)

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 77. 2. An isolated polypeptide comprising the amino acid sequence, wherein the polypeptide differs from the amino acid sequence of SEQ ID NO: 2 or 77 at amino acid 125, amino acid 138 and amino acid 193, and the polypeptide has cell division Synthetic activity. 3. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3 or 76. 4. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) encoding comprises the nucleotide sequence of the aminoacid sequence of SEQ ID NO:2 or 77; (b) an isolated polypeptide encoding the following amino acid sequence, wherein the polypeptide differs from the amino acid sequence of SEQ ID NO: 2 or 77 at amino acid 125, amino acid 138 and amino acid 193, and the polypeptide has a cytokinin synthetic activity. 5. A transgenic plant cell comprising a polynucleotide operably linked to a promoter that initiates expression in a plant, wherein the polynucleotide contains the nucleotide sequence of claim 4, wherein the cell in the plant The levels of mitogens are modulated compared to control plants. 6. The plant cell of claim 5, wherein the level of said cytokinin is increased. 7. The plant cell of claim 5, wherein said polynucleotide is operably linked to a tissue-biased promoter, a constitutive promoter, or an inducible promoter. 8. The plant cell of claim 7, wherein the promoter is a root-biased promoter, a leaf-biased promoter, a shoot-biased promoter or an inflorescence-biased promoter. 9. The plant cell of claim 5, wherein said modulation of cytokinin levels affects flower development. 10. The plant cell of claim 5, wherein said modulation of cytokinin levels affects root development. 11. The plant cell of claim 5, wherein said plant has an altered ratio of roots to roots. 12. The plant cell of claim 5, wherein the seed size or seed weight is increased. 13. The plant cell of claim 5, wherein the vigor or biomass production of said plant is increased. 14. The plant cell of claim 5, wherein said plant has increased stress tolerance. 15. The plant cell of claim 5, wherein said plant is maize and has reduced apical kernel abortion. 16. The plant cell of claim 5, wherein said promoter is stress-insensitive and is expressed in developing seed or related maternal tissue at or near anthesis. 17. The plant cell of claim 5, wherein said plant is corn, wheat, rice, barley, sorghum, or rye. 18. A plant cell genetically engineered at a locus of a native genome comprising the polynucleotide of claim 4, wherein the plant's cytokinin levels are regulated. 19. A method of modulating cytokinin levels in a plant comprising transforming said plant with the polynucleotide of claim 4 operably linked to a promoter. 20. The method of claim 19, wherein said modulation of cytokinin levels affects root growth or root ratio. 21. The method of claim 19, wherein said modulation of cytokinin levels affects flower development. 22. The method of claim 19, wherein said modulation of cytokinin levels increases seed size or seed weight. 23. The method of claim 19, wherein said modulation of cytokinin levels increases plant stress tolerance. 24. The method of claim 19, wherein said modulation of cytokinin levels affects viability or biomass production. 25. The method of claim 19, wherein said operably linked promoter is a tissue-biased and/or inducible promoter. 26. The method of claim 19, wherein the promoter is stress-insensitive and is expressed in developing seed or related maternal tissue at or near anthesis. 27. The method of claim 19, wherein senescence is delayed. 28. The method of claim 19, wherein the sink strength of the plant seed is modulated. 29. The method of claim 28, wherein cytokinin levels are increased in one or more of the embryo, endosperm, and tissues proximal thereto. 30. The method of claim 29, wherein said proximal tissue comprises a pedicel. 31. A method of modulating the rate or incidence of shoot regeneration in callus comprising expressing in said callus the polynucleotide of claim 4 operably linked to a heterologous promoter. 32. The method of claim 31, wherein said promoter is inducible.

Images