CN101292027A - Ribulose-1,5-bisphosphate carboxylase/oxygenase activating enzymes with improved thermostability and methods of use thereof - Google Patents

Abstract

The present invention provides thermostable polypeptides related to Arabidopsis Rubisco Activase polypeptides. Nucleic acids encoding the polypeptides of the invention are also provided. Methods for using the polypeptides and nuclei acids of the invention to enhance resistance of plants to heat stress are encompassed.

Description

The ribulose-1,5-bisphosphate that thermostability improves, 5-bisphosphate carboxylase/oxygenase activating enzymes and using method thereof

Invention field

The present invention relates generally to the photosynthesis level of raising growing plants under the intensification condition.In particular, the present invention relates to improve the thermostability that photosynthetic enzyme is the Rubisco activating enzymes.

Background of invention

Ribulose-1,5-bisphosphate, 5-bisphosphate carboxylase/oxygenase (Rubisco) are a kind of important enzymes in the photosynthesis process.This kind of enzyme in the photosynthesis process with CO ₂Be incorporated in the plant.Atmosphericoxygen and CO ₂Competition causes photorespiration as the substrate of Rubisco, makes Rubisco become rate-limiting step in the photosynthesis.

Rubisco activating enzymes (RCA) are the activatory protein of a kind of catalysis Rubisco, and the activation of Rubisco is regulated photosynthesis by causing photosynthetic carbon reduction and photorespiration oxidation of coal.Rubisco activating enzymes catalysis ribulose-1,5-bisphosphate, 5-bisphosphate (RuBP) is from the release of Rubisco.On the Rubisco newly do not occupy the position now with regard to empty can be in conjunction with CO ₂And Mg ²⁺Activator so that photosynthesis carry out.The Rubisco photosynthetic enzyme also is responsible for discharging sugar-fbpase inhibitor from Rubisco, thereby recovers the Rubisco catalytic activity.Therefore, if the Rubisco activating enzymes are impaired, Rubisco keeps non-activity, and photosynthesis slows down.

The Rubisco activating enzymes are to thermally labile, and therefore its activity of rising along with temperature reduces.As a result, the photosynthesis process slows down owing to the Rubisco activatory lacks.The Rubisco activating enzymes are the meeting sex change under the intensification condition, thereby makes it non-activity Rubisco can not be changed into activity form.Arabidopis thaliana (Arabidopsis) contains two kinds of RCA isotypes (isoform)---short thermolability isotype (RCA1) and long relatively hot stability isotype (RCA2), they produce (Werneke etc. by the alternately montage of pre-mRNA, 1989, Plant Cell 1:815-825).

The growing crop plant can be had benefited from the raising of photosynthesis level in megathermal climate.Therefore, if can make the rate-limiting step in the photosynthesis more heat-resisting, crop plant can more easily be grown in these weathers.

Summary of the invention

The present invention relates to than the Rubisco activating enzymes that natural Rubisco activating enzymes the have more thermostability polypeptide of deriving.Rubisco activating enzymes of the present invention are derived in the plant of polypeptide under being grown in the intensification condition to a great extent, and (substantially) keeping activity.The nucleic acid molecule of code book invention polypeptide is also contained in the present invention.

Except Rubisco activating enzymes of the present invention are derived the polypeptide, will be appreciated that also the present invention also contains this variant polypeptides, include but not limited to any similar to a great extent sequence of this polypeptide, any fragment, analogue, homologue, mutant or modified polypeptide.The variant that the present invention is contained has functionally active (being one or more known function activity that they can show wild-type Rubisco activating enzymes) in the intensification condition on small part.The nucleic acid molecule of the variant polypeptide of encoding is also contained in the present invention.

The carrier that comprises one or more nucleic acid of the present invention is also contained in the present invention.

The cell that comprises polypeptide of the present invention, nucleic acid molecule and/or carrier is also contained in the present invention.

The invention still further relates to the transgenic plant that comprise polypeptide of the present invention, nucleic acid molecule and/or carrier.These transgenic plant can be with any way express transgenic well known in the art, and described mode includes but not limited to constitutive expression, grows and regulate expression, tissue specific expression etc.The seed that obtains from transgenic plant of the present invention is also contained in the present invention.

The accompanying drawing summary

The sign of Figure 1A-1C: wild-type RCA (RCA1) and thermostability variant (183H12,301C7 and 382D8).(A) the Rubisco activity after after 25 ℃ (whites), 40 ℃ (grey) and 45 ℃ (black) are handled down, being activated enzyme activation.Activating enzymes albumen incubation under assigned temperature was measured under 25 ℃ after 15 minutes.(B) activation of Rubisco under the catalytic condition of 25 ℃ (whites) and 40 ℃ (grey).(C) the proteic atpase activity of the activating enzymes that incubation was measured under 25 ℃ after 15 minutes under assigned temperature.

Fig. 2 A-2D:Rubisco activating enzymes mutant strain (Δ rca) is at environment CO ₂Under sign.(A) immunoblotting assay of the leaf of Arabidopis thaliana wild-type (RCA/RCA), heterozygosis (RCA/ Δ rca) and (the Δ rca/ Δ rca) plant of isozygotying.Trace carries out immunity with anti-reorganization Arabidopis thaliana RCA1 polyclonal antibody and decorates (immunodecorated).(B) with the fluoroscopic image analysis measure three age in week wild-type (on) and the photosynthesis performance of Δ rca (descending) plant.(C) (B) described plant (every kind of phenotype 50 strain plant) at the leaf area of specifying under age in week.(D) eight age in week wild-type (on) and the photo of Δ rca (descending) plant.

Fig. 3 A-3C: the characterization of molecules of Δ rca mutant strain.(A) the allelic synoptic diagram of wild-type (RCA) and absence type (Δ rca).Each RCA exon of each numeral.Indicated forward and reverse primer, be respectively RCAf and RCAr and rcaf and rcar in order to amplification RCA and the allelic 1.4kb PCR of Δ rca product.(B) pcr analysis of the T1 plant of expression 183H12 (RCA primer-last little figure; Rca primer-following little figure).Strain system numbering (top) and genetic background (below) have been indicated.(C) (B) described in the western blot analysis of gross protein (5 μ g/ swimming lane) of leaf of each strain system.Trace is surveyed with anti-reorganization RCA1 polyclonal antibody.

Fig. 4 A-4D: each Δ rca mutant strain of expressing RCA1 and thermostability variant 183H12,301C7 and 382D8 the having complementary functions of (22 ℃) under the normal growth condition.The strain of each each independent transformation event of numeral is a label.(A) three age in week leaf the immunoblotting assay of gross protein.(B) each photo shows that all above-mentioned plant size when growing under normal operation is similar.(C) the photosynthesis performance of fluoroscopic image analysis above-mentioned each plant of monitoring out (8-10 strain plant/independent strain system).(D) temporary transient (1 hour) moderate-heat stress be handled the influence to the photosynthesis speed of the Δ rca transgenic line of expressing RCA1 and thermostability variant.The net photosynthesis that every kind of strain is four strain independence plant is to monitor under 22 ℃ (whites) and 30 ℃ (grey) with infrared gas analyzer.

Fig. 5 A-5F: moderate-heat stress (every day 30 ℃ handle 4 hours) to expressing RCA1 or the wild-type plant of thermostability variant 183H12,301C7 and 382D8 and the influence of Δ rca mutant strain.(A) photo of each plant shows the difference speed of growth of RCA variant mediation.(B) the every strain that analyzes with the fluoroscopic image analytical system is the leaf area of 8-10 strain independence plant.Band same letter person does not reach significant difference (P=0.05) for adopting protection least significant difference method (LSD) behind the mean number.(C) at the net photosynthesises of four strain independence plant of the 30 ℃ of selected strain of each of monitoring out by the gaseous interchange analysis after following 2 hours systems.(D) photo of the ripe plant described in (C) (8 age in week).(E) the silique quantity of every strain plant.Each strain system has analyzed 8-10 strain independence plant.(F) from selected strain be seed weight/1000 seed of the seed of (each strain is 5 strain independence plant) results.

Fig. 6 A-6D:26 ℃ heat stress is to expressing wild-type plant and the growth of Δ rca mutating strain series and the influence of output of RCA1 and thermostability variant 183H12.(A) the silique quantity of every strain plant.Each strain system has analyzed 10-12 strain independence plant.(B) photo of the silique of the selected strain of each of growing under assigned temperature system has shown the variation of silique size and setting percentage.Rod=0.5cm.(C) seed weight/1000 seed of the seed of each plant results of describing in (A).(D) percentage of germination (22 ℃) of the seed (each strain is 250 seeds) of each plant results of the Arabidopis thaliana of growth under 22 ℃ (whites) or 26 ℃ (grey).

Detailed Description Of The Invention

The invention provides the polypeptide that spreads out from the Rubisco activating enzymes. The present invention also provides the nucleic acid molecules of code book invention polypeptide. The stable on heating method that improves plant with polypeptide of the present invention and nucleic acid is also contained in the present invention, and described method comprises that strengthening Rubisco activates Thermostability.

Polypeptide of the present invention

The present invention relates to than the Rubisco activating enzymes that natural Rubisco activating enzymes the have more heat endurance polypeptide of deriving. In preferred embodiments, the Rubisco activating enzymes polypeptide of deriving is among the SEQ ID NO:4,6,8,10,12,14,16,18,20 and 22 any. Polypeptide of the present invention is also contained the those polypeptides by any Rubisco activating enzymes derivative nucleic acids coding of the present invention.

Except Rubisco activating enzymes of the present invention are derived the polypeptide, will be appreciated that also the present invention also contains the variant of this polypeptide, any to a great extent similar sequence, any fused polypeptide, any fragment, analog, homologue, mutant or the modified polypeptide that includes but not limited to this polypeptide. The variant that the present invention is contained is at least part of under Elevated Temperature Conditions to have a functional activity (being that they can show that one or more known functions of wild type Rubisco activating enzymes are active). This functional activity includes but not limited to: biologically active, such as the activation to Rubisco; Antigenicity, namely in conjunction with or with wild type Rubisco activating enzymes (including but not limited to SEQ ID NO:2) the competitions ability in conjunction with anti-Rubisco activating enzymes antibody; Immunogenicity, namely producing can be in conjunction with the ability of the antibody of wild type Rubisco activating enzymes polypeptide. In preferred embodiments, variant has at least a functional activity that is similar to a great extent or is better than its parent's polypeptide (be unaltered Rubisco activating enzymes derive polypeptide). Just as used herein, if the functional activity of variant within a standard deviation of parent's polypeptide, thinks that then variant " is similar to " parent's polypeptide to a great extent.

In one embodiment, such polypeptide is contained in the present invention, it has at least a functional activity (for example Rubisco activation) of Rubisco activating enzymes under Elevated Temperature Conditions, and with any peptide sequence among the SEQ ID NO:4,6,8,10,12,14,16,18,20 and 22 at least 85%, 90%, 95%, 97%, 98% or 99% homogeneity is arranged. In specific embodiment, this peptide species of the present invention carries out the best when comparing as its sequence and SEQ ID NO:2, has more than one in corresponding to the residue 42,130,131,168,257,274 of SEQ ID NO:2,293 and 310 position, more than two, more than five or seven above positions change. Just carry out the amino acid sequence that the best is compared with reference sequences, certain amino acid " corresponding to " in the reference sequences with this residue in that position of comparing middle pairing.

Just as used herein, with certain sequence definition for to have with reference sequences in the situation of " X% identity at least ", for example " certain polypeptide and SEQ ID NO:4 have at least 95% identity " will be appreciated that " X% identity " is meant absolute identity percentage ratio, unless otherwise.Term " absolute identity percentage ratio " is meant the sequence identity percentage ratio of measuring like this: identical amino acid or nucleic acid (nucleic acid) are counted 1 fen, any displacement are counted 0 fen, and no matter the similarity of mispairing amino acid or nucleic acid how.In typical sequence alignment, " the absolute identity percentage ratio " of two sequences is represented with the percentage ratio of amino acid or nucleic acid " identity ".In the bests of two sequences comparison manipulation require sequence or insert in the situation in spaces two sequences simultaneously therein, for the purpose of measuring identity percentage ratio, with in the sequence with another sequence in space paired amino-acid residue think mispairing.The space can be an internal voids, or external void, i.e. brachymemma.Can for example use Clustal W program (version in June, 1.8,1999) easily to determine absolute identity percentage ratio (Thompson etc., 1994, Nucleic Acids Research 22:4673-4680) with default parameters.

In another embodiment, the present invention is contained and is comprised the derive fusion polypeptide of polypeptide or its variant of Rubisco activating enzymes.In a specific embodiment, with certain peptide (as U.S. Patent Application Serial 11/150, those disclosed peptide in 054) be added on the polypeptide of the present invention, this peptide instructs its polypeptide that connects to navigate to plant plastid (plalstid) or photosynthesis of plant organ in view of the above.

In another embodiment, the derive fragment of polypeptide of Rubisco activating enzymes is contained in the present invention.Such polypeptide is contained in the present invention, it has at least a functionally active (for example Rubisco activation) of Rubisco activating enzymes under the intensification condition, and is at least 25,50,75,100,125,150,175,200,225,250,275,300,325,350,375 or 380 continuous amino acids in any length among the SEQ ID NO:4,6,8,10,12,14,16,18,20 and 22.In preferred embodiments, the segmental polynucleotide of coded polypeptide can be under stringent condition with coding SEQ ID NO:4,6,8,10,12,14,16,18,20 and 22 in any nucleic acid hybridization.

In a specific embodiment, fragment of the present invention is corresponding to certain functional domain of Rubisco activating enzymes, include but not limited to ATP in conjunction with territory and substrate interaction territory (referring to for example Li etc., (2005) J Biol Chem 280:24864-24869; Salvucci etc., (1994) Biochemistry 33:14879-14886 and van de Loo etc., (1996) Biochemistry35:8143-8148).

In another embodiment, polypeptide analog is contained in the present invention.Polypeptide analog can have adorned residue, promptly by modifying to the Rubisco activating enzymes polypeptide of deriving the molecule of any kind is covalently bound.Polypeptide analog of the present invention can be such as but not limited to the derivatize by glycosylation, acetylize, PEGization, phosphorylation, amidation, known protection/blocking groups, proteolysis cutting, modify with proteinic be connected etc. of cell ligand or other.Polypeptide analog of the present invention can be modified by chemically modified with the technology that well known to a person skilled in the art, described technology includes but not limited to that the metabolism of specificity chemical chop, acetylize, formylation, tunicamycin is synthesized.In addition, the analogue of polypeptide of the present invention can contain an above atypia amino acid.

In another embodiment of the invention, the Rubisco activating enzymes polypeptide of deriving is not SEQ ID NO:2.In yet another embodiment of the present invention, the Rubisco activating enzymes polypeptide of deriving is not natural wild type peptide.

The present invention also provides the method that for example produces polypeptide of the present invention by recombinant means.

Nucleic acid molecule of the present invention

The invention still further relates to the derive nucleic acid molecule of polypeptide of coding Rubisco activating enzymes.In preferred embodiments, Rubisco activating enzymes derivative nucleic acids molecule is any among the SEQ ID NO:3,5,7,9,11,13,15,17,19 and 21.Nucleic acid molecule of the present invention is also contained derive those nucleic acid molecule of polypeptide of coding any Rubisco activating enzymes of the present invention.

Except coding Rubisco activating enzymes are derived the nucleic acid molecule of polypeptide, will be appreciated that nucleic acid molecule of the present invention also contains derive those nucleic acid molecule of variant polypeptides polypeptide of coding Rubisco activating enzymes, described variant includes but not limited to Rubisco activating enzymes derive any similarly sequence, any fusion polypeptide, any fragment, analogue, homologue, mutant or the modified polypeptide to a great extent of polypeptide.The such polypeptide of nucleic acid molecule variant coding that the present invention is contained, it has functionally active (being one or more known function activity that they can show wild-type Rubisco activating enzymes) in the intensification condition on small part.

In one embodiment, such nucleic acid molecule is contained in the present invention, and any in itself and SEQ IDNO:3,5,7,9,11,13,15,17,19 and 21 the nucleic acid molecule has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity.In specific embodiment, the such polypeptide of this nucleic acid molecule encoding of the present invention, its nucleotides sequence broomrape and SEQ ID NO:2 carry out the best when comparison, have more than one in the position corresponding to the residue 42,130,131,168,257,274,293 of SEQ IDNO:2 and 310, more than two, more than five or seven above positions change.

For measuring the identity percentage ratio of two nucleic acid molecule, with two sequences for the best comparison purpose compare (for example, can in the sequence of first nucleic acid molecule, introduce the space) so that carry out the best comparison with second nucleic acid molecule.Nucleotide to the corresponding nucleotide position compares then.When certain position in first sequence was occupied by the Nucleotide identical with the Nucleotide of corresponding position in second sequence, then two molecules were identical in this position.Identity percentage ratio between two sequences is the function (being % identity=identical lap position number/total positional number x100%) of the common same position number of these two sequences.In one embodiment, two sequence lengths are identical.

The also available mathematical algorithm of mensuration of the identity percentage ratio between two sequences is finished.A limiting examples of mathematical algorithm that is used for the comparison of two sequences, be algorithm (Karlin and the Altschul of Karlin and Altschul, (1990) Proc.Natl.Acad.Sci.87:2264-2268, at Karlin and Altschul, done correction among (1993) Proc.Natl.Acad.Sci.90:5873-5877).This algorithm is incorporated into (Altschul etc., (1990) J.Mol.Biol.215:403 and Altschul etc., (1997) Nucleic Acid Res.25:3389-3402) in NBLAST and the XBLAST program.Can openly obtain in order to the software that carries out the BLAST analysis, for example obtain by American National biotechnology information center (National Center forBiotechnology Information).This algorithm relates to the short word (short word) that at first is tested and appraised the length W in the search sequence (query sequence) and identifies that high sub-sequence is to (high scoring sequence pair, HSP), described short word when with database sequence in equal length word when comparison coupling or satisfy a certain positive threshold value (positive-valued threshold score) T.T is called neighborhood word threshold value (Altschul etc., the same).These initial neighborhood word are hit (heighborhood word hit) and are served as the seed of searching the search of the longer HSP that contains them in order to startup.Then word is hit and extend to accumulation comparison mark (cumulative alignment score) along each sequence to both direction and can farthest be increased.The calculating of running summary of the points scored is to use parameter M (the prize branch of a pair of coupling residue for nucleotide sequence; Always＞0) and the N (point penalty of mispairing residue; Always＜0).For polypeptide, use the matrix (scoring matrix) of keeping the score to calculate running summary of the points scored.The extension that word hits on each direction is interrupted when following situation occurring: accumulation comparison mark departs from its maximum implementation value (maximum achieved value) and reaches numerical value X; Running summary of the points scored divides the accumulation of residue comparison to become below 0 or 0 because of negative more than one; Perhaps reached the end of arbitrary sequence.BLAST algorithm parameter W, T and X have determined the sensitivity and the speed of comparison.BLASTN program (for nucleotide sequence) is used word length (W) 11, expected value (E) 10, cutoff value 100, M=5, N= ^-Article 4 and two, the comparison of chain is as default setting.For polypeptide, the BLASTP program use word length (W) 3, expected value (E) 10 and BLOSUM62 keep the score matrix as default setting (referring to Henikoff ﹠amp; Henikoff, (1989) PNAS 89:10915).

Also can use Clustal V comparison method to determine identity percentage ratio (Higgins and Sharp, (1989) CABIOS 5:151-153), this method is present in the LASERGENE information biology and calculates bag (bioinformatics computing suite) (DNASTAR Inc., Madison is in Megalign program WI)." default parameters " is the parameter that the program manufacturer presets, they are corresponding to space point penalty (GAP PENALTY)=10 and gap lengths point penalty (GAP LENGTH PENALTY)=10 for multisequencing comparison, and they are KTUPLE 1, space point penalty=3, WINDOW=5 and DIAGONALS SAVED=5 for two sequence alignments.After using Clustal V program to carry out the comparison of sequence, check " sequence distance (sequence the distance) " form on this program, can obtain " identity percentage ratio ".

Identity percentage ratio between two sequences can be used and above-mentioned technology similar techniques, is allowing or is not allowing to measure under the space.When calculating identity percentage ratio, only accurate coupling is counted usually.

In another embodiment, the fragment of Rubisco activating enzymes derivative nucleic acids molecule is contained in the present invention.Such nucleic acid molecule is contained in the present invention, it has at least a functionally active (for example Rubisco activation) of Rubisco activating enzymes under the intensification condition, and is at least 100,250,500,750,950,1000 or 1100 continuous nucleotides in any length among the SEQ ID NO:3,5,7,9,11,13,15,17,19 and 21.

In a specific embodiment, fragment of the present invention is corresponding to the nucleic acid molecule of the functional domain of coding Rubisco activating enzymes, described functional domain include but not limited to ATP in conjunction with territory and substrate interaction territory (referring to for example Li etc., (2005) J Biol Chem 280:24864-24869; Salvucci etc., (1994) Biochemistry 33:14879-14886 and van de Loo etc., (1996) Biochemistry 35:8143-8148).

In another embodiment, the present invention contain can be under stringent condition with SEQ IDNO:3,5,7,9,11,13,15,17,19 and 21 in the nucleic acid molecule of any hybridization.Word " stringent condition " be meant certain nucleic acid can be with its target nucleic acid hybridization in the complex mixture that is present in nucleic acid usually and basically not can with the condition of other nucleic acid hybridization.Stringent condition has sequence dependent, will be different under different situations.Long nucleic acid especially will be hybridized under comparatively high temps.This area relevant for the detailed guidance of nucleic acid hybridization (Tijssen for example, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, " Overview of principles of hybridization and the strategyof nucleic acid assays " (1993)).Usually, high stringent condition is chosen to be than the heat fusion joint (T of specific nucleic acid under the ionic strength pH that determines _m) low about 5-10 ℃.Low stringency condition is chosen to be usually and compares T _mLow about 15-30 ℃.T _mBe that temperature (under ionic strength, pH and the nucleic acid concentration determined) when being in the hybridization balance of 50% target nucleic acid complementary probe and target nucleic acid is (because the excessive existence of target nucleic acid, at T _mHaving 50% probe to be in down occupies in the balance).The condition that hybridization conditions is normally such: under pH7.0-8.3 salt concn less than about 1.0M Na ion concentration, be generally about 0.01-1.0M Na ion concentration (or other salt), temperature is at least about 30 ℃ for short probe (for example 10-50 Nucleotide), is at least about 60 ℃ for long probe (for example greater than 50 Nucleotide).Stringent condition also can be realized by adding destabilizing agent such as methane amide.For selectivity or specific hybrid, positive signal doubles background hybridization at least, preferred 10 times to background hybridization.Word " specific hybrid " is meant when specific nucleotide sequence is present in the complex mixture (for example total cell or library DNA or RNA), and certain molecule combines with this sequence following of stringent hybridization condition, two strandsization or hybridize.

In another embodiment of the invention, Rubisco activating enzymes derivative nucleic acids molecule is not SEQ ID NO:1.In yet another embodiment of the present invention, Rubisco activating enzymes derivative nucleic acids molecule is not natural wild-type nucleic acid molecule.

The carrier that comprises nucleic acid molecule of the present invention is also contained in the present invention.Cell or the plant that comprises carrier of the present invention also contained in the present invention.

Term " nucleic acid molecule " in this article refers to from 5 ' terminal strand or dichain polymer to 3 ' terminal that read, deoxyribonucleotide or ribonucleotide based composition.It comprises the DNA or the RNA of chromosomal DNA, self-replacation plasmid and main execution architecture effect.

Rubisco activating enzymes derived sequence

Rubisco activating enzymes of the present invention are derived polypeptide and nucleic acid molecule can be by introducing an above residue displacement, adding and/or disappearance produce in wild-type (wt) Rubisco activating enzymes (including but not limited to Arabidopis thaliana Rubisco activating enzymes (SEQ ID NO:2)).Usually, produce the Rubisco activating enzymes polypeptide of deriving,, perhaps unfavorable characteristic is reduced so that the appropriate characteristics of wild-type Rubisco activating enzymes polypeptide is strengthened.In one embodiment, the derive thermostability of polypeptide of Rubisco activating enzymes has raising than wild-type Rubisco activating enzymes.In another embodiment, the Rubisco activating enzymes polypeptide of deriving is similar to or is higher than the enzymic activity of wild-type Rubisco activating enzymes under normal condition (for example 25 ℃) in the enzymic activity under the intensification condition (for example 40 ℃).

In one embodiment, produce Rubisco activating enzymes derivative nucleic acids molecule with wild-type Rubisco activating enzymes nucleic acid molecule (for example SEQID NO:1) as template.In some embodiments, nucleic acid residue change with coding such amino-acid residue more than, make changed by coded amino acid, described amino-acid residue when nucleotide sequence is carried out best comparison with SEQ IDNO:2 discovery corresponding to the residue 42,130,131,168,257,274,293 and 310 of SEQ ID NO:2.

Can be by standard techniques calling sequence change (sequence alteration), described technology such as directed molecular evolution technology, for example DNA reorganization method (referring to for example Christians etc., (1999) Nature Biotechnology 17:259-264; Crameri etc., (1998) Nature391:288-291; Crameri etc., (1997) Nature Biotechnology 15:436-438; Crameri etc., (1996) Nature Biotechnology 14:315-319; Stemmer, (1994) Nature 370:389-391; Stemmer etc., (1994) Proc.Natl.Acad.Sci.91:10747-10751; United States Patent (USP) 5,605,793,6,117,679,6,132,970,5,939,250,5,965,408,6,171,820; International prospectus WO 95/22625, WO 97/0078, WO 97/35966, WO 98/27230, WO 00/42651 and WO 01/75767); Site-directed mutagenesis (referring to for example Kunkel, (1985) Proc.Natl.Acad.Sci.82:488-492; Oliphant etc., (1986) Gene 44:177-183); Oligonucleotide directed mutagenesis (referring to for example Reidhaar-Olson etc., (1988) Science 241:53-57); Chemomorphosis (referring to for example Eckert etc., (1987) Mutat.Res.178:1-10); Fallibility PCR is (referring to for example Caldwell ﹠amp; Joyce, (1992) PCR Methods Applic.2:28-33) and cassette mutagenesis (referring to for example Arkin etc., (1992) Proc.Natl.Acad.Sci., 89:7871-7815); (mainly referring to for example Arnold, (1993) Curr.Opinion Biotechnol.4:450-455; Ling etc., (1997) Anal.Biochem., 254 (2): 157-78; Dale etc., (1996) Methods Mol.Biol.57:369-74; Smith, (1985) Ann.Rev.Genet.19:423-462; Botstein etc., (1985) Science, 229:1193-1201; Carter, (1986) Biochem.J.237:1-7; Kramer etc., (1984) Cell 38:879-887; Wells etc., (1985) Gene 34:315-323; Minshull etc., (1999) Current Opinion in Chemical Biology 3:284-290).

In one embodiment, reorganize with DNA and produce Rubisco activating enzymes derivative nucleic acids molecule.DNA reorganization can external (in vitro), in vivo (in vivo), (in silico) or their combination realize on computer chip.Can realize like this at the recombination method on the computer chip: in computer, use genetic algorithm to recombinate corresponding to each sequence string (sequence string) of each homology (and even non-homogeneous) nucleic acid.The recombination sequence string option of gained for example by adopt simultaneously oligonucleotide synthetic/the gene technology (oligonucleotide synthesisgene reassembly technique) of reassemblying carries out synthetic corresponding to the nucleic acid of recombination sequence, is transformed into nucleic acid.This method can produce at random, the part at random or the design change.Many detail content of relevant reorganization on computer chip, comprise the uses in computer system such as genetic algorithm, genetic operator, and the generation of the combination of the nucleic acid of corresponding nucleic and design (for example selecting (cross-over site selection)) based on the exchange site, also have design, recombination method pseudo-random or at random, description (referring to for example international prospectus WO 00/42560 and WO 00/42559) is all arranged in the art.

In another embodiment, adopt directed mutagenesis, change, produce Rubisco activating enzymes derivative nucleic acids molecule by selecting specific nucleotide sequence or position in the wild-type Rubisco activating enzymes.This orthomutation can be introduced any position in nucleic acid, can be conservative sudden change or non-conservative sudden change.

" non-conservative amino acid replacement " is that wherein amino-acid residue is had the metathesis of certain radical amino acid replacement of dissimilar side chain.The existing in the art definition of family with amino-acid residue of similar side chain.These families comprise amino acid with basic side chain (Methionin for example, arginine, Histidine), amino acid (aspartic acid for example with acid side-chain, L-glutamic acid, l-asparagine, glutamine), amino acid (glycine for example with uncharged polar side chain, Serine, Threonine, tyrosine, halfcystine), amino acid (L-Ala for example with non-polar sidechain, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), amino acid (Threonine for example with β-branched building block, Xie Ansuan, Isoleucine) and amino acid (tyrosine for example with aromatic side chains, phenylalanine, tryptophane, Histidine).

This orthomutation can not be non-conservative amino acid residues displacement, but the conservative amino acid residues displacement; Perhaps this orthomutation can also be the conservative amino acid residues displacement except being the non-conservative amino acid residues displacement." conservative amino acid replacement " is that wherein amino-acid residue is had the metathesis of certain radical amino acid replacement of similar side chain.After mutagenesis, it is recombinant expressed carry out by coded protein, and can measure this activity of proteins.

In some embodiments, can make displacement, make the amino acid at 42 places, position have uncharged polar side chain or β-branched building block, the amino acid at 130 places, position has basic side chain, the amino acid at 131 places, position has non-polar sidechain or β-branched building block, the amino acid at 168 places, position has basic side chain, the amino acid at 257 places, position has non-polar sidechain or β-branched building block, the amino acid at 274 places, position has basic side chain, and the amino acid that the amino acid at 293 places, position has basic side chain and/or 310 places, position has acid side-chain.Amino acid position can carry out the best comparison and determines by derive amino acid sequence of polypeptide and SEQ ID NO:2 of the Rubisco activating enzymes that will be encoded.

In another embodiment, produce Rubisco activating enzymes derivative nucleic acids molecule with random mutagenesis.Can on the whole piece chain of encoding sequence or a part of chain, introduce sudden change (for example passing through saturation mutagenesis) at random.In certain embodiments, coding can be had analog structure territory, structural motif, avtive spot, the nucleotide sequence that other related polypeptide of mispairing or imperfect coupling is arranged when perhaps comparing with the part of wild-type Rubisco activating enzymes is used for mutagenic processes to produce the diversity of sequence.

It should be understood that for each the mutagenesis step in some above-mentioned technology, can carry out arbitrary or repeatedly recirculation in steps so that the diversity optimization of sequence.Above-mentioned each method can be used in combination with any suitable order.In many cases, aforesaid method produces nucleotide sequence or a collection of recombinant host cell that comprises the nucleotide sequence of change that a collection of (a pool of) changes.Have the nucleotide sequence of change of desired characteristic or the host cell of the nucleotide sequence that express to change, can identify by screening with one or more mensuration well known in the art.Mensuration can be carried out under the condition of the polypeptide that can select to have required physics or chemical property.The change of nucleotide sequence can be checked order to determine by the nucleic acid molecule of polypeptide that the coding among the clone is changed.

In addition, Rubisco activating enzymes derivative nucleic acids molecule can on the whole or carry out codon optimized on the part.Because any one amino acid (except that methionine(Met)) all is coded by a plurality of codons (table 1), the sequence of nucleic acid molecule can be changed and coded amino acid is changed.When an above codon changes on nucleic acid level to meet or during better near the codon usage of specific host, it is codon optimized that Here it is.The frequency of the preferred codon usage that host cell is shown, the mean value of frequency that can be by obtaining the preferred codon usage in the expressed a large amount of genes of this host cell calculates.This analysis can be defined in this host cell the gene of highly expressing.United States Patent (USP) 5,824,864 for example provide the frequency of the codon usage of the shown highly expressing gene of dicotyledons and monocotyledons.Those of ordinary skills will appreciate that have the form of the preference information that multiple biology is provided and other bibliography to obtain in this area.

Measure the method for Rubisco activated enzyme activity

The present invention relates to compare Rubisco activating enzymes that thermostability the improves polypeptide of deriving with wild-type Rubisco activating enzymes.Term used herein " thermostability improves " is meant that the ability of deriving described Rubisco activating enzymes polypeptide comparing activation Rubisco under the intensification condition with wild-type Rubisco activating enzymes improves.In one embodiment, the Rubisco activating enzymes derive polypeptide in the enzymic activity of (for example 35 ℃ or higher) under the intensification condition greater than the enzymic activity of wild-type Rubisco activating enzymes under the intensification condition.In another embodiment, derive polypeptide under the intensification condition (for example 26 ℃ or higher of Rubisco activating enzymes, for more preferably 40 ℃ of external tests) enzymic activity be similar to a great extent or be higher than (for example 20-25 ℃ under normal operation of wild-type Rubisco activating enzymes, for more preferably 25 ℃ of external tests, for measuring more preferably 22 ℃ in the body) enzymic activity.Term used herein " is similar to a great extent " and is meant that the Rubisco activating enzymes derive the enzymic activity of polypeptide within a standard deviation of the enzymic activity of wild-type Rubisco activating enzymes.

Available any method well known in the art is measured the Rubisco activating enzymes activity (include but not limited to Rubisco activation and ATP hydrolysis) of polypeptide under the intensification condition of deriving.

In some embodiments, the Rubisco activating enzymes polypeptide active of deriving is measured external.In one embodiment, can measure the Rubisco activating enzymes derive polypeptide in the source that comprises the Rubisco of inactivation, RuBP, ATP and mark carbon ([C for example ¹⁴] NaHCO ₃) solution in the ability of its activation Rubisco during incubation.But the mix situation of monitoring mark carbon in 3-phoshoglyceric acid (GPA) is as Rubisco activatory sign.In another embodiment, can measure the Rubisco activating enzymes polypeptide ability of its hydrolysising ATP during incubation in comprising the solution of ATP of deriving.Before measuring, the Rubisco activating enzymes polypeptide of deriving is heat-treated, then under the intensification condition or measure under normal operation.Can use the Rubisco activation of purifying to use composition, also can use to comprise the cell of Rubisco activation with composition.

In other embodiment, the Rubisco activating enzymes polypeptide active of deriving is measured in vivo.Can make the plant (for example deletion mutantion strain) of not expressing wild-type Rubisco activating enzymes express one or more Rubisco activating enzymes polypeptide of deriving, and can analyze photosynthesis speed, biomass, the speed of growth and the seed production of this plant under the intensification growth conditions.This plant can grow under the intensification condition fully, perhaps can grow the short period under the intensification condition.In one embodiment, photosynthesis speed is by analyzing the CO of plant ₂Fixedly measure.In another embodiment, the speed of growth is to measure by the leaf area of analyzing plant.In another embodiment, seed production is to analyze by the seed weight of measuring ripe dried plant.In another embodiment, seed production is to measure by measuring rate of emergence.

Strengthen the stable on heating method of plant

The Rubisco activating enzymes can activate the Rubisco in the plant.Activatory Rubisco participates in photosynthesis, and it is the rate-limiting step in the photosynthesis process.Along with the decline of Rubisco activated enzyme activity, Rubisco keeps non-activity, and photosynthesis slows down or stops.The rising of temperature can make Rubisco activating enzymes instability and/or sex change, thereby makes this enzyme not too can maybe the Rubisco of non-activity can not be changed into activity form.The present invention relates to compare Rubisco activating enzymes that thermostability the improves polypeptide of deriving with wild-type Rubisco activating enzymes.Thus, under the Rubisco activating enzymes that thermostability improves were derived the participation of polypeptide, the photosynthesis process can become and have more thermotolerance.

Available any method well known in the art is impelled expression of plants one or more Rubisco activating enzymes of the present invention polypeptide of deriving.In one embodiment, can make up the transgenic plant that to express one or more polypeptide of the present invention.These transgenic plant can be expressed one or more polypeptide of the present invention (for example overall situation expression) in a organized way in institute.Perhaps, one or more polypeptide of the present invention can only be expressed (for example tissue specific expression) in a part (a subset of) tissue, preferably express in participating in photosynthetic tissue or organoid (for example plastid).Polypeptide of the present invention can be in plant constitutive expression, perhaps under the control of inducible promoters, express.In some embodiments, the expression of the endogenous Rubisco activating enzymes of plant and/or activity are lowered or eliminate.

Recombinant expressed

Nucleic acid molecule of the present invention and polypeptide can with the recombinant DNA of standard well known in the art and molecule clone technology (Sambrook for example, Fritsch and Maniatis, Molecular Cloning:A Laboratory ManualCold Spring Harbor Laboratory Press:Cold SpringHarbor, (1989)) carry out recombinant expressed.In addition, can use recombinant DNA technology to produce the nucleic acid construct thing that is suitable for making up transgenic plant.

Therefore, one aspect of the present invention relates to the carrier that comprises nucleic acid molecule of the present invention or its variant, the preferred expression carrier.Term used herein " carrier " is meant the polynucleotide that can transport another nucleic acid that has been connected thereto.One type carrier is " plasmid ", and it is meant the circular double-stranded DNA ring that wherein can introduce other DNA section.The carrier of another kind of type is a virus vector, and wherein other DNA section can be incorporated in the viral genome.

Some carrier can carry out self-replicating (bacteria carrier and the episomal vector that for example have the bacterium replication orgin) in the host cell that they were incorporated into.Other carrier (for example non-add type carrier) is incorporated in the genome of host cell in being incorporated into host cell the time, thereby along with host genome is duplicated.In general, the expression vector that is applicable to recombinant DNA technology often is the form of plasmid (carrier).But the present invention has a mind to comprise the expression vector of other form, as virus vector (for example replication defect type retrovirus).

Recombinant expression vector of the present invention comprises the nucleic acid molecule of the present invention that its form is suitable for its expression in host cell.This means that this recombinant expression vector comprises more than one the adjusting sequence of selecting according to the host cell of waiting to be used for expressing, described adjusting sequence combines (operably associated) with polynucleotide generation operability to be expressed.In the middle of recombinant expression vector, " operability in conjunction with " is meant that the purpose nucleotide sequence is connected (described expression for example in in-vitro transcription/translation system, perhaps when carrier is introduced in the host cell in host cell) in the mode that can make it and expressed with the adjusting sequence.Term " adjusting sequence " means and comprises promotor, enhanser and other expression controlling elements (for example polyadenylation signal).This adjusting sequence have in the art description (Goeddel for example, Gene Expression Technology: Methods in Enzymology, (1990) Academic Press, San Diego, CA).Regulate that adjusting sequence that sequence comprises that those instruct nucleotides sequence to be listed in to carry out constitutive expression in polytype host cell and those instruct that nucleotide sequence only expresses in some host cell adjusting sequence (for example tissue specificity adjusting sequence).The design that those of skill in the art will recognize that expression vector must be depended on such as following factor; Need position of expressing etc. in the selection of host cell to be transformed, required protein expression level, the organism.Expression vector of the present invention can be incorporated in the host cell, thereby produce coded protein of nucleic acid molecule as herein described or peptide, comprise fusion rotein and peptide.

In some embodiments, the isolating nucleic acid that serves as promotor or enhancer element can be incorporated into the appropriate location (normally upstream) of the non-allos form (non-heterologous form) of polynucleotide of the present invention, to regulate the expression of polynucleotide of the present invention up or down.For example, can carry out changing in the body (referring to United States Patent (USP) 5,565,350 to endogenesis promoter by sudden change, disappearance and/or displacement; International patent application no PCT/US93/03868), perhaps isolating promotor can be incorporated in the vegetable cell with correct direction with from the correct distance of associated gene (cognate gene) of polynucleotide of the present invention, with the expression of controlling gene.Genetic expression is adjusted being suitable for obtaining under the condition of plant-growth, with change polypeptide of the present invention in vegetable cell total concn and/or change its composition.Therefore, the invention provides natural, endogenous (the being non-allos) allogeneic promoter of form and/or the composition of enhanser and the method that is connected to (operably linked to) polynucleotide of the present invention in order to the preparation operability.

Carry out expression of polypeptides if desired, need comprise the polyadenylation zone at the 3 ' end in polynucleotide encoding district usually.Can spread out from natural gene in this polyadenylation zone, spread out from multiple other plant gene or spread out from T-DNA.3 ' end sequence to be added can spread out from for example nopaline synthase or octopine synthase gene, perhaps spreads out from another plant gene, does not perhaps more preferably spread out from any other eukaryotic gene.

Recombinant expression vector of the present invention can be designed to can (enterobacteriaceae (Enterobacteriaceae) for example be as Escherichia (Escherichia) at prokaryotic cell prokaryocyte; Bacillaceae (Bacillaceae); Rhizobiaceae (Rhizoboceae) belongs to (Rhizobacter) as rhizobium (Rhizobium) and bacillus radicicola; Spirillaceae (Spirillaceae) is as photogenic bacterium (photobacterium); Zymomonas (Zymomonas); Serratia (Serratia); Aeromonas (Aeromonas); Vibrio (Vibrio); Desulfovibrio (Desulfovibrio); Spirillum (Spirillum); Lactobacillaceae (Lactobacillaceae); Pseudomonadaceae (Pseudomonadaceae) is as pseudomonas (Pseudomonas) and genus acetobacter (Acetobacter); Azotobacteraceae (Azotobacteraceae) and Nitrobacteraceae (Nitrobacteraceae)) or eukaryotic cell (for example insect cell (use rhabdovirus expression vector), yeast cell, vegetable cell or mammalian cell) in express polypeptide of the present invention (discussion of relevant proper host cell referring to Goeddel, the same).Perhaps, can for example use T7 promotor adjusting sequence and T7 polysaccharase at in-vitro transcription and translation recombinant expression vector.

The expression of protein in prokaryotic organism, but often with comprising the composing type that instructs fusion rotein or non-Expression of Fusion Protein or the carrier of inducible promoter, in intestinal bacteria, carry out.Fusion vector is added to a plurality of amino acid therein in the encoded protein matter, normally is added to the N-terminal of recombinant protein.This fusion vector plays following at least three purposes usually: 1) increase the expression of recombinant protein; 2) solvability of increase recombinant protein; And/or 3) help purifying recombinant proteins by the part that serves as in the affinity purification.Usually, in fusion expression vector, the proteolysis cleavage site is incorporated into the contact (junction) that merges part (fusion moiety) and recombinant protein, so that recombinant protein can be separated with the fusion part after the purifying of fusion rotein.This kind of enzyme and their related (cognate) recognition sequence comprise factor Xa, zymoplasm and enteropeptidase.Typical fusion expression vector comprises pGEX (Pharmacia BiotechInc; Smith and Johnson, (1988) pMAL (New EnglandBiolabs Gene 67:31-40),, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), they respectively with glutathione S-transferase (GST), maltose E is conjugated protein or A albumen is fused to the target recombinant protein.

In another embodiment, expression vector is a Yeast expression carrier.Example in order to the carrier of expression in yeast saccharomyces cerevisiae (S.cerevisiae) comprises pYepSecl (Baldari etc., (1987) EMBO is J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) pJRY88 (Schultz etc. Cell30:933-943),, (1987) pYES2 (Invitrogen Corp. Gene 54:113-123),, San Diego, CA) and pPicZ (Invitrogen Corp., San Diego, CA).

Perhaps, expression vector is a rhabdovirus expression vector.Can comprise pAc series (Smith etc. for the baculovirus vector of marking protein in the insect cell of cultivating (for example Sf 9 cells), Mol.Cell Biol.3:2156-2165) and pVL series (Lucklow and Summers, (1989) Virology 170:31-39) (1983).

In another embodiment, use plant expression vector, include but not limited to tobacco mosaic virus (TMV) and potato virus expression vector, nucleic acid molecule of the present invention is expressed in vegetable cell.

Other is used for prokaryotic cell prokaryocyte and eukaryotic appropriate expression system is well known in the art (referring to for example Sambrook etc., (1990) Molecular Cloning, A Laboratory Manual, the 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, the 16th among NY and 17 chapters).

There is multiple promotor to can be used for enforcement of the present invention.Can select promotor according to required result.But nucleic acid and composing type, tissue-specific induction type or other promotor can be made up, in host living beings, to express.

" tissue-specific promoter " can instruct the expression of nucleic acid of the present invention in particular organization, organ or cell type.But tissue-specific promoter can be an induction type.Similarly, tissue-specific promoter only the certain time-frame (time frame) in this tissue or start in the etap transcribe.Can all there be activity in other tissue-specific promoter in the whole life of particular organization.Those of ordinary skills will appreciate that tissue-specific promoter can drive the expression of sequence in the tissue beyond the target tissue that is effectively connected.Therefore, tissue-specific promoter used herein is such promotor, and it preferentially drives in target tissue or cell type and expresses, but also can cause certain expression in other tissue.There is multiple tissue-specific promoter to can be used for the present invention.The all available suitable promoter targeting of any organ, these organs are as (shoot vegetative) organ/structure (for example leaf, stem and stem tuber) of sprouting, root, flower and floral organ/structure (for example bract, sepal, petal, stamen, carpel, pollen sac and ovule), seed (comprising plumule, endosperm and kind skin) and fruit.For example, instruct expression promoter and/or the photosynthesis organ specific promoters (as Khoudi etc., in (1997) Gene 197:343 disclosed RBCS promotor) of nucleic acid molecule in leaf to can be used for strengthening photosynthesis.In addition, can obtain tissue specific expression by such peptide is joined polypeptide of the present invention, described Toplink instructs connected polypeptide to navigate to photosynthesis organ (for example those disclosed organ in the U.S. Patent Application Serial 11/150,054).

" constitutive promoter " be defined as can instruct gene the expression in a organized way, promoters active under most of envrionment conditionss and growth or cytodifferentiation state.The example of constitutive promoter comprises cauliflower mosaic virus (CaMV) 35S transcription initiation zone, spread out from 1 ' of the T-DNA of Agrobacterium tumefaciems (Agrobacterium tumafaciens)-or 2 '-promotor and other transcription initiation zone from various plant genes known to a person of ordinary skill in the art.This gene for example comprises the ACT11 (Huang etc. from Arabidopis thaliana; (1996) Plant Mol.Biol.33:125-139); Cat3 (GenBank accession number U43147 from Arabidopis thaliana; Zhong etc.; (1996) Mol.Gen.Genet.251:196-203); gene (Genbank accession number X74782 from the coding stearyl-acyl carrier protein desaturase of rape (Brassica napus); Solocombe etc.; (1994) Plant Physiol.104:1167-1176); GPc1 (GenBank accession number X15596 from corn; Martinez etc.; (1989) J.Mol.Biol.208:551-565) with from Gpc2 (the GenBank accession number U45855 of corn; Manjunath etc., (1997) Plant Mol.Biol.33:97-112).Any strong constitutive promoter such as CaMV 35S promoter all can be used for the expression of polypeptide of the present invention in whole strain plant.

Term " but inducible promoter " is meant the promotor that is under accurate environment or the growth control.But the example that can influence the envrionment conditions of transcribing of inducible promoter comprises the spray of executing of the existence of anoxic condition, high temperature, light or chemical/hormone.

The suitable constitutive promoter that is used for plant host cell comprises for example core promoter (international prospectus WO99/43838 and United States Patent (USP) 6,072,050) of Rsyn7 promotor and other relevant components type promotor; Core CaMV 35S promoter (Odell etc., (1985) Nature 313:810-812); Rice actin promotor (McElroy etc., (1990) Plant Cell 2:163-171); Ubiquitin promotor (Christensen etc., (1989) Plant Mol.Biol.12:619-632 and Christensen etc., (1992) Plant Mol.Biol.18:675-689); PEMU (Last etc., (1991) Theor.Appl.Genet.81:581-588); MAS (Velten etc., (1984) EMBO are J.3:2723-2730); ALS promotor (United States Patent (USP) 5,659,026) and other (for example United States Patent (USP) 5,608,149,5,608,144,5,604,121,5,569,597,5,466,785,5,399,680,5,268,463,5,608,142 and 6,177,611).

Another aspect of the present invention relates to the host cell of having introduced recombinant expression vector of the present invention.Term " host cell " and " recombinant host cell " can be used in this article mutually.It should be understood that this term not only refers to specific experimenter's cell, also refer to the filial generation or the potential filial generation of this cell.Because because the cause of sudden change or environmental influence some variation may occur in follow-up each generation, this daughter cell in fact may be inequality with parental cell, but they still are included in the scope of this term used herein.

Therefore, the invention provides host cell with the expression vector that comprises nucleic acid molecule of the present invention or its variant.Host cell can be any eukaryotic cell (for example intestinal bacteria, bacillus thuringiensis (Bacillus thuringiensis) or eukaryotic cell (for example insect cell, yeast or vegetable cell).Thereby the present invention also provides and expresses the method that nucleic acid molecule of the present invention prepare encoded polypeptide, and described method comprises i) under the condition that encoded polypeptide is produced cultivation comprise the cell of nucleic acid molecule of the present invention; Ii) separate expressed polypeptide.

Carrier DNA can be incorporated in prokaryotic cell prokaryocyte or the eukaryotic cell by the conversion or the rotaring dyeing technology of routine.Term used herein " conversion " and " transfection " mean multiple art-recognized in order to the foreign nucleus acid molecule is incorporated into the technology in the host cell, comprise transfection, lipofection, electroporation, Agrobacterium tumefaciems (Agrobacterium tumefaciens) and the vacuum infiltration of calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-dextran.The suitable conversion or the method for transfection host cell (for example Sambrook etc. is the same) are arranged in this area.

The generation of transgenic plant

Any method well known in the art be can adopt, plant or vegetable cell transformed with nucleic acid molecule of the present invention.Nucleic acid molecule can be incorporated in the DNA of plants (for example genomic dna or chloroplast DNA), perhaps keep not being inserted into (for example by using any karyomit(e)) in the DNA of plants.The suitable method that nucleic acid molecule is incorporated in the vegetable cell comprises microinjection (Crossway etc., (1986) Biotechniques 4:320-334); Electroporation (Riggs etc., (1986) Proc.Natl.Acad.Sci.83:5602-5606; D ' Halluin etc., (1992) Plant Cell4:1495-1505); Agrobacterium-mediated conversion (United States Patent (USP) 5,563,055 and 5,981,840; Osjoda etc., (1996) Nature Biotechnology 14:745-750; Horsch etc., (1984) Science 233:496-498; Fraley etc., (1983) Proc.Natl.Acad.Sci.80:4803 and Gene Transfer-Plants, Potrykus, (editor), Springer-Verlag, Berlin 1995); Direct gene shifts (Paszkowski etc., (1984) EMBO are J.3:2717-2722); The trajectory particle quickens (ballistic particle acceleration) (United States Patent (USP) 4,945,050,5,879,918,5,886,244,5,932,782; Tomes etc., (1995) " Direct DNA Transfer intoIntact Plant Cells via Microprojectile Bombardment, Plant Cell, Tissue and Organ Culture:Fundamental Methods, Gamborg and Phillips (editor), Springer-Verlag, Berlin and McCabe etc., (1988) Biotechnology 6:923-926); Virus-mediated conversion (United States Patent (USP) 5,889,191,5,889,190,5,866,785,5,589,367 and 5,316,931); Pollen transforms (De Wet etc., (1985) The Experimental Manipulation of Ovule Tissues, Chapman etc. (editor), Longman, New York, 197-209 page or leaf); Lec 1 transforms (U.S. Patent Application Serial 09/435,054; International prospectus WO 00/28058); Whisker mediated transformation (whisker-mediatedtransformation) (Kaeppler etc., (1990) Plant Cell Reports 9:415-418; Kaeppler etc., (1992) Theor.Appl.Genet.84:560-566); The and chloroplast(id) transforms technology (Bogorad, (2000) Trends in Biotechnology 18:257-263; Ramesh etc., (2004) Methods Mol Biol.274:301-7; Hou etc., (2003) Transgenic Res.12:111-4; Kindle etc., (1991) Proc.Natl.Acad.Sci.88:1721-5; Bateman and Purton, (2000) Mol Gen Genet.263:404-10; Sidorov etc., (1999) Plant are J.19:209-216).

Be used to produce the conversion Scheme Selection of transgenic plant and vegetable cell, can be according to becoming as the plant that transforms target or the type (being monocotyledons or dicotyledons) of vegetable cell.Be particularly suitable for the example of the conversion scheme of specified plant type, comprise the conversion scheme that is suitable for following plant: potato (Tu etc., (1998) Plant Molecular Biology 37:829-838; Chong etc., (2000) Transgenic Research 9:71-78); Soybean (Christou etc., (1988) Plant Physiol.87:671-674; McCabe etc., (1988) BioTechnology 6:923-926; Finer and McMullen, (1991) In Vitro Cell Dev.Biol.27P:175-182; Singh etc., (1998) Theor.Appl.Genet.96:319-324); Corn (Klein etc., (1988) Proc.Natl.Acad.Sci.85:4305-4309; Klein etc., (1988) Biotechnology6:559-563; Klein etc., (1988) Plant Physiol.91:440-444; Fromm etc., (1990) Biotechnology 8:833-839; Tomes etc., (1995) " Direct DNA Transfer intoIntact Plant Cells via Microprojectile Bombardment, " Plant Cell, Tissue With Organ Culture:Fundamental Methods, Gamborg (editor), (Springer-Verlag, Berlin)); Cereal (Hooykaas-Van Slogteren etc., (1984) Nature 311:763-764; United States Patent (USP) 5,736,369).

In some embodiments, use more than one construction to be converted and produce transgenic plant and vegetable cell.A plurality of constructions can be introduced in cis or trans position.In preferred embodiments, each construction all has promotor and other adjusting sequence.

Transgenic plant can be by any mode express transgenic well known in the art, and described mode includes but not limited to constitutive expression, grows to regulate and express and tissue specific expression.In a specific embodiment, use can instruct the expression promoter (as Khoudi etc., in Gene 197:343 disclosed RBCS promotor) of nucleic acid molecule in leaf and/or photosynthesis organ to express nucleic acid molecule of the present invention and/or polypeptide.

The transformed plant cells that obtains by any above-mentioned transformation technology can be cultivated, have required phenotypic whole strain plant thereby to have the transforming gene type to regenerate.This regeneration techniques depends on the manipulation to the certain plants hormone in the tissue culture growth substratum, and described manipulation depends on biocide and/or the weedicide mark of having introduced with required nucleotide sequence usually.Existing in the art description of plant regeneration of carrying out from the protoplastis of cultivating (Evans etc. for example, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, 124-176 page or leaf, MacMillilan Publishing Company, New York, 1983; And Binding, Regeneration of Plants, Plant Protoplasts, 21-73 page or leaf, CRC Press, Boca Raton, 1985).Also can obtain regeneration from plant callus, explant, organ or their part (part).This regeneration techniques also has description (for example Klee etc., (1987) Ann.Rev.of Plant Phys 38:467-486) in the art.

Term " plant " comprises whole strain plant, (shoot vegetative) organ/structure of sprouting (for example leaf, stem and stem tuber), root, flower and floral organ/structure (for example bract, sepal, petal, stamen, carpel, pollen sac and ovule), seed (comprising plumule, endosperm and kind skin) and fruit (ripe ovary), plant tissue (for example vascular tissue, standard weave etc.) and cell (for example guard cell, ovum, algal filament (trichome) etc.) and their filial generation.The classification that can be used for the plant of the inventive method comprises the of all categories of the high and lower plant that is suitable for transformation technology, comprises angiosperm (monocotyledons and dicotyledons), gymnosperm, pteridophyte and many cells algae.The present invention also comprises the plant of a plurality of ploidy levels, comprises aneuploid, polyploid, amphiploid, monoploid and hemizygote plant.

Nucleic acid molecule of the present invention can be used to required proterties is imparted to any basically plant.Therefore, the present invention can be used for various plants, comprises each kind of following each platymiscium: Agrotis, allium (Allium), Ananas (Ananas), Anacardium (Anacardium), apium (Apium), Arachis (Arachis), Asparagus (Asparagus), Athamantha, Atropa (Atropa), Avena (Avena) le Sinobambusa (Bambusa), Beta (Beta), Btassica (Brassica), Brome (Bromus), Browaalia, Camellia (Camellia), Cannabis (Cannabis), papaya belongs to (Carica), Ceratonia (Ceratonia), olecranon Macroptilium (Cicer), Chenopodium (Chenopodium), Chicorium, Citrus (Citrus), Citrullus (Citrullus), Capsicum (Capsicum), safflower belongs to (Carthamus), cocoanut (Cocos), Coffea (Coffea), Coix (Coix), Cucumis (Cucumis), Cucurbita (Cucurbita), Cynodon (Cynodon), orchardgrass (Dactylis), Datura (Datura), Daucus (Daucus), Carnation (Dianthus), Digitalis (Digitalis), Wild yam (Dioscorea), oil palm belongs to (Elaeis) Finger-millet and belongs to (Eliusine), Euphorbia (Euphorbia), festuca (Festuca), Ficus (Ficus), Fragaria (Fragaria), Geranium (Geranium), Glycine (Glycine), Gramineae (Graminae), Gossypium (Gossypium), Helianthus (Helianthus), (Heterocallis), Hevea (Hevea), hibiscus (Hibiscus), Hordeum (Hordeum), poison tobacco (Hyoscyamus), sweet potato genus (Ipomoea), Lactuca (Lactuca), Lathyrus (Lathyrus), Lens culinaris belongs to (Lens), lilium (Lilium), linum (Linum), lolium (Lolium), Lotus (Lotus), lupinus (Lupinus), tomato belongs to (Lycopersicon), Queensland nut belongs to (Macadamia), Seven barks (Macrophylla), Malus (Malus), the fruit that dies belongs to (Mangifera), cassava (Manihot), Majorana, Medicago (Medicago), Musa (Musa), Narcissus (Narcissus), Nemesia, Nicotiana (Nicotiana), donkey Macroptilium (Onobrychis), Olea (Olea), (Olyreae), Oryza (Oryza), Panicum (Panicum), Panicum (Panicum), (Panieum), Pennisetum (Pannisetum), Pennisetum (Pennisetum), green winter Solanum (Petunia), Pelargonium (Pelargonium), Persea (Persea), (Pharoideae), Phaseolus (Phaseolus), ladder forage spp (Phleum), Picea (Picea), annual bluegrass belongs to (Poa), Pinus (Pinus), Pistachia, Pisum (Pisum), Populus (Populus), Pseudotsuga (Pseudotsuga), pear (Pyrus), Prunus (Prunus), Pseutotsuga, Psidium (Psidium), oak belongs to (Quercus), Ranunculus (Ranunculus), Rhaphanus (Raphanus), currant belongs to (Ribes), Ricinus (Ricinus), cuckoo belongs to (Rhododendron), rose (Rosa), saccharum (Saccharum), beauty's flap belongs to (Salpiglossis), Secale (Secale), Senecio (Senecio), setaria (Setaria), sequoia sempervirens belongs to (Sequoia), sinapsis alba belongs to (Sinapis), Solanum (Solanum), sorghum (Sorghum), Herba Stenotaphri helferi belongs to (Stenotaphrum), Theobromus, Semen Trigonellae belongs to (Trigonella), Clover (Trifolium), Semen Trigonellae belongs to (Trigonella), Triticum (Triticum), Hemlock (Tsuga), Tulipa (Tulipa), Vetch (Vicia), Vitis (Vitis), Vigna (Vigna) and Zea (Zea).

In specific embodiment, transgenic plant are corn, tomato, potato, paddy rice, soybean, cotton, Sunflower Receptacle, clover, lettuce, canola oil dish, Chinese sorghum or tobacco plant.

But the render transgenic plant-growth is also with same transformed plant or different plant pollinations.Can grow for two generations or how for plant, to guarantee that required nucleic acid molecule, polypeptide expression and/or phenotype feature obtain stablely keeping and following.Those of ordinary skills will appreciate that, nucleic acid molecule of the present invention be stabilized be incorporated in the transgenic plant and be identified can operate (operable) after, can it be incorporated in other plant by sexual hybridization.Can use any in the breeding technique of multiple standards, this depends on the kind of generation hybridization.

The mensuration of the expression in the transgenic plant

Can use any method well known in the art, measure nucleic acid molecule of the present invention or by the expression level of its encoded polypeptides in plant.For example, the expression level useful molecules technology of nucleic acid molecule encoded polypeptide of the present invention in plant measured, and described technology includes but not limited to immunoassay, immuno-precipitation, gel electrophoresis and quantitatively gel electrophoresis.

In addition, the expression level of nucleic acid molecule encoded polypeptide of the present invention in plant can be measured by the change degree of comparing plant phenotype (including but not limited to photosynthesis speed, the many and seed production of growth) under the intensification condition with the plant of expressing wild-type Rubisco activating enzymes.

In addition, extract or the polypeptide from transgenic plant and separate tissue thereof can be used for external test.

The content of the paper of all publication that this paper quoted from, books, reference manual and digest integral body by reference is attached to herein, more fully to describe the state-of-art in field under the present invention.

Owing to can not depart from scope and spirit of the present invention various variations made in above-mentioned theme, therefore stipulate to be comprised in all above descriptions and/or appended claims defined in theme all should be interpreted as being intended to describing and explanation the present invention.Can make various modifications and variations to the present invention according to above instruction content.

Embodiment

Embodiment 1: separate the Rubisco activating enzymes polypeptide of deriving

With the wild-type Rubisco activating enzymes of SEQ ID NO:1 as template, from single-gene reorganization (single gene shuffling) (referring to for example Crameri etc., (1998) Nature.391 (6664): 288-91; Chang etc., (1999) Nat Biotechnol.17 (8): 793-7; Ness etc., (1999) Nat Biotechnol.17 (9): 893-6; Christians etc., (1999) NatBiotechnol.17 (3): 259-64 and US Patent Nos.6,605,430,6,117,679; And5,605,793) and synthetic reorganization (synthetic shuffling) (referring to for example US Patent No.6,436,675 and international prospectus WO 00/42561, WO 01/23401, WO00/42560; And WO 00/42559) produces Rubisco activating enzymes library.

Briefly, use

Reagent is pressed the greenery isolation of RNA of the scheme (Invitrogen) of manufacturer from Arabidopis thaliana.Use TITANIUM ^TMOne-step RT-PCR Kit (BDBiosciences-Clontech) is cloned into RCA cDNA (GenBank accession number NM179990) PCR

In the carrier (Invitrogen).Reorganize for single-gene, in the first round, ripe RCA short-form (coding region V59-K438) is carried out pcr amplification (Qiagen Taq archaeal dna polymerase or Stratagene Mutazyme archaeal dna polymerase) in no primer PCR reaction, fragmentation and reassemblying is saved the gene of being reorganized with the flank primer that contains NcoI site (5 ') and BamHI site, 6x-His coding region and terminator codon (3 ') then.With the library clone of variant to the coli expression carrier of NcoI and BamHI digestion (pET16b, Novagen).Be to enlarge the hereditary storehouse (pool of genetic) of the first round, synthesize reorganization (referring to Ness etc., (2002) NatBiotechnol.20:1251-1255) with the diversity of wheat, paddy rice, cotton, spinach and cucumber.By before Crameri etc., the description of (1998) Nature 15:288-291 is carried out second gene of taking turns with first round variant as the parent and is reorganized.

The Rubisco activating enzymes that thermostability improves are derived polypeptide by following screening method separation.

The first step: Rubisco activation determinationDerive the colibacillary culture of polypeptide at 4 ℃, 3 with expressing the Rubisco activating enzymes, centrifugation after 15 minutes under the 500rpm ,-80 ℃ of storages down in PCR plate at the bottom of the 96 hole V-arrangements.Prepare the Bacillus coli cells split product like this: culture was at room temperature thawed 5 minutes, add the ultrasonic damping fluid of 75 μ l (100mMTricine KOH pH8.0,20mM xitix, 3mM Mg-ATP, 10mM MgCl to every hole ₂, 10%v/v glycerine, 10mM β me, x3.33 proteinase inhibitor, 1 μ l/ml benzonase, 1mg/ml N,O-Diacetylmuramidase), plate is 4 ℃ of following joltings 60 minutes, cleaved up to cell precipitation.Plate was carried out supersound process 1 minute with MISONIX microplate ultrasonoscope, cooled off then 1 minute.This process repeats four times.With culture at 4 ℃, 4, under the 000rpm centrifugal 20 minutes.The supernatant liquor that will contain soluble protein is used for the Rubisco activation determination.

The intestinal bacteria supernatant liquor of 22 μ l is transferred to flicker plate (scintillation-plate) at the bottom of the 96 hole U-shapeds, at room temperature the incubation 15 minutes split product of (and in mensuration, be used as " normal condition ").The thermal treatment of split product is by the intestinal bacteria supernatant liquor of 35 μ l being transferred at the bottom of the 96 hole V-arrangements PCR plate and being carried out in 15 minutes at 40 ℃ of following incubations.Then heat treated supernatant liquor is transferred to flicker plate at the bottom of the 96 hole U-shapeds split product of (and in mensuration, be used as " heat-treat condition ").Two plates before carrying out performance measurement 4 ℃ of following incubations 5 minutes.

Measure Rubisco activation situation like this: will contain Rubisco activating enzymes or Rubisco activating enzymes and derive the Arabidopis thaliana Rubisco (15 μ g) of inactivation of the product of cell lysis of polypeptide and purifying at reaction buffer (100mM Tricine KOH pH8.0,10mM MgCl ₂, 10mM[ ¹⁴C] NaHCO ₃, Mg-ATP, 4mM RuBP, 1mM PEP, 40 μ g/ml pyruvate kinases) in incubation 15 minutes under the room temperature (referring to Shen etc., (1991) J. Biol.Chem.266:8963-8968).Add 1N HCl and stop expressing the Rubisco activating enzymes and derive the product of cell lysis of polypeptide, measure by the liquid-scintillation spectrometry method to the activation of Rubisco ¹⁴CO ₂The situation of mixing.

Rubisco used in the said determination obtains from Arabidopis thaliana leaf purifying.Leaf homogenized and freezing in liquid nitrogen, be suspended in again then and extract damping fluid (100mM Hepes-KOHpH8.0,1mM EDTA pH8.0,3mM DDT, 0.5mM PMSF, 10mM MgCl ₂, 10mM NaHCO ₃) in.Suspension at 4 ℃, 12, under the 000rpm centrifugal 20 minutes, is collected supernatant liquor.Supernatant liquor is remained on ice under continuously stirring, add ammonium sulfate to ultimate density simultaneously and be 35% saturated, at 4 ℃, 12, under the 000rpm centrifugal 20 minutes.With supernatant liquor restir 30 minutes, add ammonium sulfate to ultimate density simultaneously and be 55% saturated, then at 4 ℃, 12, under the 000rpm centrifugal 20 minutes.The gained precipitation is dissolved in the extraction damping fluid, precipitates also centrifugal again with 18% polyoxyethylene glycol.Again gained precipitation is suspended in again and extracts in the damping fluid (the original 40ml supernatant liquor of about 1ml/), at 4 ℃, 13, under the 000rpm centrifugal 30 minutes.The Rubisco of purifying is in supernatant liquor, and adding glycerine to ultimate density is 10%.

Make the Rubisco inactivation (referring to Wang etc., (1992) PlantPhysiol.100:1858-1862) of purifying by following scheme.Add 10mM DTT to the Rubisco of purifying, 45 ℃ of following incubations 10 minutes.The gained mixture is got 1ml to be added to level pad (50mMTricine-KOH pH8.0 and 0.5mM EDTA pH8.0) equilibrated 20ml SephadexG-50 post.The level pad that adds 1ml/ part elutes the Rubisco of inactivation from post.The inactivation Rubisco that collection elutes, at room temperature incubation is 1 hour, then in the presence of 4mMRuBp incubation on ice 1 hour.

The second stage: the HTP temperature overview of active clone's product of cell lysis activation Rubisco (temperature profile)The purpose that identifies in the first step screening is cloned in the screening of the second stage and does further to characterize.Product of cell lysis of each active clone is remake test as mentioned above, exception be under four different temperature (16 ℃, 25 ℃, 40 ℃ and 45 ℃), to carry out Temperature Treatment earlier before measuring.Select the clone who compares thermostability overview with wild-type Rubisco activating enzymes (SEQ ID NO:2) and carry out third stage screening with relative raising.

The third stage: the temperature overview of the Rubisco activating enzymes variant activation Rubisco of purifyingFor measuring the specific activity of the polypeptide of deriving that front two-stage screening identified, the polypeptide of affinity purification is carried out preincubation under differing temps, analyze their then 25 ℃ of abilities of activation Rubisco down.Because the Rubisco activating enzymes are with the Rubisco of time-dependent manner mode catalysis inactivation, to each reaction with three minutes time interval monitorings 15 minutes.The ratio of Rubisco activating enzymes: Rubisco is set at 1: 40, is similar to the ratio in the plant leaf.The derive thermostability of polypeptide of wild-type Rubisco activating enzymes (SEQ ID NO:2) and Rubisco activating enzymes is displayed in Table 2.Thermostability percentage ratio is represented under 40 ℃ and is accounted under 25 ℃ by the percentage ratio of the amount of Rubisco activating enzymes activatory Rubisco by the amount of Rubisco activating enzymes activatory Rubisco.

The derive vitro characterization of polypeptide of embodiment 2:Rubisco activating enzymes

The polypeptide of deriving of isolating Rubisco activating enzymes among the embodiment 6.1 is carried out in vitro tests in three different mensuration, to measure their specific activity and thermostabilitys (t.s.) under 25 ℃ and 40 ℃.In all situations, all will be set at 100% 25 ℃ of results that obtain down with wild-type Rubisco activating enzymes.

The activation of inactivation RubiscoThe Rubisco activating enzymes of the purifying polypeptide of deriving is measured described mensuration by embodiment 1 first step, exception be 40 ℃ or 45 ℃ of following incubations 15,30,45 or 60 minutes at the polypeptide that carries out will deriving earlier before the Rubisco activation performance is measured.The result shows at the 2-4 of table 3 row.The 2nd row representative of table 3 is in the amount of the activation Rubisco that inactivation Rubisco and Rubisco activating enzymes are obtained behind 25 ℃ of following incubations.Do not use the intensification condition.After being listed as 40 ℃ of thermal treatments representing 15 minutes, the 3rd of table 3 accounted under 25 ℃ by the percentage ratio of the amount of Rubisco activating enzymes activatory Rubisco by the amount of Rubisco activating enzymes activatory Rubisco.The 4th row of table 3 are represented after 40 ℃ of thermal treatments of 45 minutes by the amount of Rubisco activating enzymes activatory Rubisco and are not accounted for when heat-treating (under 25 ℃) by the percentage ratio of the amount of Rubisco activating enzymes activatory Rubisco.

The derive Rubisco activation of polypeptide 301C7 and 382D8 of Rubisco activating enzymes demonstrates high thermostability (Figure 1A) under 40 ℃ and 45 ℃ of processing.The RCA1 that 382D8 handles under uniform temp in the specific activity after 45 ℃ of processing is high by 80%, than in the activity of the RCA1 of 25 ℃ of following incubations only low 10%.

Rubisco activation under catalytic conditionThe Rubisco activating enzymes of the purifying polypeptide of deriving is measured described mensuration by embodiment 1 first step, exception be that to measure be to carry out (Crafts-Brandner and Salvucci, (2000) PNAS97:13430-13435) under intensification condition (promptly 40 ℃).The result shows at the 5-6 of table 3 row.The 5th row representative of table 3 is in the amount of the activation Rubisco that inactivation Rubisco and Rubisco activating enzymes are obtained behind 25 ℃ of following incubations.Do not use the intensification condition.The 6th row representative of table 3 is accounted for when measuring under 25 ℃ by the percentage ratio of the amount of Rubisco activating enzymes activatory Rubisco by the amount of Rubisco activating enzymes activatory Rubisco when measuring for 40 ℃.

Wild-type RCA keeps down 0.5 Rubisco active state at 40 ℃, and derive polypeptide 183H12,301C7 and 382D8 of Rubisco activating enzymes can keep the active state (Figure 1B) of 0.62-0.72 under the same conditions.With respect to the reaction under 25 ℃, the thermostability variant is 40 ℃ of Rubisco active state that keep 78-98% down, and the enzyme of wild-type keeps 70%.The protein that all shows maximum specific activity under 25 ℃ or 40 ℃ is at isolating best variant 183H12 of the first round.

Atpase activityBecause the Rubisco activating enzymes are that the bonded ATP enzyme that needs ATP to unclamp Rubisco and sugar-phosphoric acid (contains AAA in the polypeptide ⁺Structural domain), therefore tested the influence of temperature to the ATP hydrolytic action of Rubisco activating enzymes.The intrinsic activity of monitoring activating enzymes mixture and no matter itself and the interactional atpase assay of Rubisco often are used to Rubisco activating enzymes sign.Atpase assay has been carried out in description by Salvucci ((1992) Arch.Biochem.Biophys.298:688-696).The result shows at the 7-8 of table 3 row.The amount of the 7th row representative existing hydrolysising ATP behind 25 ℃ of following incubations of table 3 with Rubisco activating enzymes and ATP.Do not use the intensification condition.The 8th row representative of table 3 with the heat treated Rubisco activating enzymes incubation through 40 ℃ after the amount of existing hydrolysising ATP enzyme, be expressed as the percentage ratio that accounts for without the amount of existing hydrolysising ATP enzyme behind the heat treated Rubisco activating enzymes incubation.

Fig. 1 C shows, the Rubisco activating enzymes derive polypeptide 301C7 and 382D8 35 ℃ and 40 ℃ down stability compare with RCA1 to have improved and surpass 10 times, and 183H12 shows 20% and 30% raising respectively under 25 ℃ and 40 ℃.

The complementation of embodiment 3:Rubisco activating enzymes deletion mutant

For in the background that disappearance is isozygotied (Δ rca/ Δ rca) (referring to Li etc., (2001) Plant is J.27:235-242) middle variant of expressing reorganization, developed following complementary cascade (complementation cascade): 1) adopt single leaf 96 hole DNA extraction method (Xin etc., (2003) BioTechniques 34:820-826), pass through HTP-PCR, with at wild-type and the allelic Auele Specific Primer of disappearance, heterozygous plant is selected at disappearance; 2) transform with goal gene; 3) T0 that carries out antibiotics resistance selects, and carries out the pcr analysis of homozygosity; 4) homozygous plants with gained carries out self-pollination, to obtain the T1 transgenic line.

Immunoblotting assay to wild-type, heterozygosis and homozygous plants (genetic background is respectively RCA/RCA, RCA/ Δ rca and Δ rca/ Δ rca) discloses, and gene product (microscler formula and short-form) is at wild-type similar with expression levels in the heterozygous plant (Fig. 2 A).Do not have short isotype and long isotype in the plant that disappearance is isozygotied, this confirms to suddenly change the expression of RCA1 and RCA2 has been eliminated.Δ rca plant is compared with wild-type, at environment CO ₂Show lower photosynthesis performance (Fq '/Fm ' value) (being respectively 0.185 ± 0.038 and 0.332 ± 0.033) (Fig. 2 B) when growing down, afterwards the leaf areas are significantly lower 3 weeks of growth on soil (is respectively 2.93 ± 0.49 and 395.4 ± 8.75mm ²) (Fig. 2 C).It is seriously short and small that two monthly age Δ rca homozygous plants and wild-type plant are compared growth, and yellows (Fig. 2 D) takes place.

Based on sequential analysis and mapping to deletion fragment, two cover primers have been designed: RCA primer (forward primer 5 '-CAGACAATGTTGGCCTC-3 ' (SEQ ID NO:23) and reverse primer 5 '-ACGAGTAACGATGGTAGG-3 ' (SEQ ID NO:24)), they are special to wild-type allele, produce the 1.5kb product; With rca primer (forward primer 5 '-GTCTATACCTTGAGC-3 ' (SEQ ID NO:25) and reverse primer 5 '-TCAGTCATACTCGG-3 ' (SEQ ID NO:26)), they produce the 1.5kb product at absence type allelotrope, produce 4.9kb product (Fig. 3 A) at wild-type allele.For with rca primer amplification 1.5kb product but the 4.9kb product that do not increase is 1.5 minutes with the pcr amplification cycle sets.The genetic background that this two covers primer is used to characterize the T1 plant.Because the conversion host is a heterozygosis for the disappearance of endogenous rca locus, expressing the genetically modified T1 plant of Rubisco activating enzymes is the mixture of wild-type (RCA/RCA), heterozygote (RCA/ Δ rca) and homozygote (Δ rca/ Δ rca).Identified the transgenic line of in different legacy backgrounds, expressing reorganization variant 183H12 with PCR screening (Fig. 3 B) and immunoblotting assay (Fig. 3 C).In containing the genetic background of at least one wild-type allele, express the plant (#13 of 183H12; Wt, #12; For disappearance is heterozygosis), have the short-form and the microscler formula of zymoprotein simultaneously.In strain is only to detect short-form among the #2 (disappearance is isozygotied), because transgenosis is designed to only express the short-form of zymoprotein.

Embodiment 4:Rubisco activating enzymes are derived, and (In planta) characterizes in the plant of polypeptide

For measuring the effect of Rubisco activating enzymes under normal temps and intensification that improves, used Arabidopis thaliana Rubisco activating enzymes deletion mutantion strain (Δ rca) (referring to embodiment 3).Deriving with wild-type Rubisco activating enzymes (SEQ ID NO:1), first round Rubisco activating enzymes, polypeptide 183H12 (SEQ ID NO:7) and two kind second are taken turns 382D8 (SEQ ID NO:15) and 301C7 (SEQ ID NO:19) has complementary functions to Δ rca.

In order in transgenic arabidopsis plant (Δ rca), to express wild-type Rubisco activating enzymes and the Rubisco activating enzymes polypeptide of deriving, with the transgenosis and two enhanser structural domain (double enhancer domain) (Day ﹠amp of the coding region of coding chloroplast transit peptides and the rca1 or the polypeptide of deriving; Maiti, (1999) Transgenics 3:61-70), UBQ3 terminator and kalamycin resistance gene nptII are together, be cloned into and contain the Mirabilis cauliflower mosaic virus (Mirabilis MosaicCaulimovirus is MMV) among the pMAXY4384 of promotor.Adopt floral dipping method (Clough etc., (1998) Plant are J.16:735-743), transform the Deleteagene of heterozygosis with Agrobacterium tumefaciems GV3101 bacterial strain ^TMThe RCA mutant strain.Express for confirming, with protein from liquid N ₂With extract in the plant tissue (2-3g fresh weight) in the extraction damping fluid (100mM Tricine-KOH pH8, EDTA pH8,10mM 2 mercapto ethanol and proteinase inhibitor cocktail Set V) of 1ml.With crude extract under 3000g centrifugal 5 minutes and 12 in succession, clarified in centrifugal 20 minutes under the 000g.The soluble protein extract of getting 10 micrograms is transferred to by (according to the specification sheets that Invitrogen provided) on the nitrocellulose filter after separating on the 10%SDS-polyacrylamide gel.The polyclonal antibody that produces with reorganization Arabidopis thaliana RCA1 carries out the immunity decoration to trace, puts together substrate reagent box (Bio-Rad) with Ap and detects protein.

Shown in Fig. 4 A, wild-type plant (RCA/RCA) is expressed the short-form and the long isotype of activating enzymes, only expresses the short isotype of 43kDa and carry out complementary transgenosis Δ rca strain system with transgenosis.(plant is at illumination in 16 hours (225 μ mol photon m under 22 ℃ of culture condition ^-2s ^-1Grow in hour dark cycle of)/8), transgenic line demonstrates the speed of growth similar to the unconverted plant of wild-type (Fig. 4 B).(FluorImager QubitSystems) analyzes the photosynthesis performance (photosynthetical system II operational efficiency Fq '/Fm ') and the speed of growth by previous description (Baker etc., (2001) J Exp.Bot.52:615-621) with the chlorophyll fluorescence imaging system.It is similar to the unconverted plant of wild-type to express the derive Fq '/Fm ' value of transgenosis disappearance strain system (being respectively Δ rcaRCA1, Δ rca183H12, Δ rca301C7 and Δ rca382D8) of polypeptide 183H12,301C7 or 382D8 of RCA1 or Rubisco activating enzymes, and the expression that shows short-form is enough under the normal growth condition to Δ rca have complementary functions (Fig. 4 C).Under these conditions, (LI6400 is Li-Cor) at 150 μ mol photon m by portable infrared gas analyzer ^-2s ^-1With 350 μ barCO ₂Photosynthesis activity and Δ rca183H12-3, Δ rca301C7-3 similar with Δ rca382D8-1 (Fig. 4 D) of the Δ rcaRCA1-1 of following measurement.Δ rcaRCA1-1 temporarily is exposed to 30 ℃ caused its photosynthesis to descend 12% in 1 hour.On the contrary, Δ rca183H12-3, Δ rca301C7-3 and Δ rca382D8-1 strain tie up to 30 ℃ and show that photosynthesis increases by 16,22 and 16% after following 1 hour.

Because being exposed to 30 ℃, the Arabidopis thaliana plant can cause the inducing on a small quantity of heat shock protein (usually 32 ℃ and above being induced) and to (the Salvucci etc. of influence slightly of stomatal aperture, (2001) Plant Physiol.127:1053-1064), carry out growth under long heat treatment with the Arabidopis thaliana plant of expressing wild-type or thermostability Rubisco activating enzymes.Will around age transgenic line be exposed to moderate heat stress two time-of-weeks.Growth conditions is illumination in 16 hours (225 μ mol photon m ^-2s ^-1) and 8 hours dark.In light circulation, plant is 22 ℃ of growths 6 hours down, 30 ℃ (2 ℃ of per minutes) growth 4 hours that is rapidly heated then, and then return 22 ℃ and grow to the light loop ends.In dark cycle, plant remains under 22 ℃.By previous description (Barth etc., (2003) Heredity.91:36-42) grow, the sign of biomass and output.Each plant shows normal phenotype and leaf color, but different sizes (Fig. 5 A).Than the unconverted plant of wild-type with express the derive Δ rca strain system of polypeptide of Rubisco activating enzymes, Δ rcaRCA1 (strain is 1,8 and 9) short and small (Fig. 5 B).Expression has, and the transgenic arabidopsis of the 183H12-3 of high external specific activity is maximum plant.The strain system of expressing 301C7 and 382D8 is bigger than Δ rcaRCA1 strain system, but does not reach the leaf area levels of 183H12 strain system.Although only expressing the Δ rca strain system of the short-form (RCA1) of wild type gene is that (expressing microscler formula and short-form simultaneously) is little than the unconverted strain of wild-type, but great majority are expressed the derive transgenic line of polypeptide of Rubisco activating enzymes and are demonstrated the leaf area bigger than the unconverted plant of wild-type (Fig. 5 B), and all are expressed Rubisco activating enzymes and derive the strain system of polypeptide all than the Δ rca transformant significantly bigger (P=0.01) of expressing RCA1.

Be exposed to two weeks moderate heat stress around age plant also demonstrate difference on the development of plants speed.When the processing phase finishes, there are 74,44 and 33% Δ rca183H12, Δ rca301C7 and Δ rca382D8 to have ripe inflorescence respectively, flower opens, and the Δ rcaRCA1 strain cording of 100% unconverted wild-type plant and 88% has the immature inflorescence in the formation, and flower does not open (data not shown).In addition, 12% Δ rcaRCA1 strain system is in the vegetative phase, does not have the visible inflorescence.Under the normal growth condition, arabidopsis thaliana is bloomed after all around.Therefore, the Δ rca183H12, the Δ rca301C7 that show normal development account for relative higher per-cent with Δ rca382D8 strain system, this is likely because due to the raising of the thermostability of RCA, this thermostability makes the photosynthesis under the moderate heat stress condition and the inhibition of growth are minimized.

The best strain system of every kind of variant is further analyzed photosynthesis activity in its stress circulate at moderate heat (30 ℃ following 2 hours after).Transgenic line demonstrates the CO relevant with the leaf area ₂Fixed mode.Δ rca183H12-3, Δ rca301C7-3 and Δ rca382D8-1 strain are CO ₂Fixed speed respectively than Δ rcaRCA1-1 strain be high by 30,25 and 23%.These results prove that the Rubisco activating enzymes are photosynthetic restrictive factors under experiment condition.

It is similar to be exposed to its outward appearance of each ripe plant (10 age in week) that moderate heat stress 8 time-of-weeks.Than Δ rcaRCA1-1, in Δ rca183H12-3, Δ rca301C7-3 and Δ rca382D8-1 strain system, detect influence (being respectively 116,121 and 119%) (Fig. 5 D) to the forward a little of plant height.Silique every strain plant quantitatively observes huge difference, Δ rca183H12-3, Δ rca301C7-3 and Δ rca382D8-1 are respectively 130.8 ± 48.2,84.3 ± 19.6 and 100.8 ± 26.9, contrast therewith, Δ rcaRCA1-1 and wild-type are respectively 40.2 ± 16.3 and 47.5 ± 15.8 (Fig. 5 E).

The relative raising that forms for silique in the transformant of the RCA that confirm to express improves is not to be that cost obtains to sacrifice single seed size, compared 1000 seeds/batch weight.Shown in Fig. 5 F, the seed that Δ rca183H12-3 and Δ rca382D8-1 produce is than Δ rcaRCA1-1 big slightly (difference big 18 and 32%), and the seed weight of Δ rca301C7-3 and wild-type plant is similar to Δ rcaRCA1-1.

Be further to analyze the RCA that improves influence to the growth under the moderate thermal stress, make the T3 strain that (external) under 25 ℃ expresses the active clone of tool be 183H12 26 ℃ and than the experiment of front continuous growth under higher light intensity and the humidity.The condition of growth is 26 ℃ and 85% humidity illumination in following 16 hours (300 μ mol photon m ^-2s ^-1) and 8 hours dark.Wild-type and Δ rcaRCA1 strain tie up to 26 ℃ and grow compared with growing under the normal growth condition down, and the total biomass of generation has decline slightly, shows that plant development speed is slower, and the constant (not shown) of biomass of each plant of Δ rca183H12 and growth course.Contrast therewith, 26 ℃ down every strain silique quantity of producing of the Δ rca plant of growth be subjected to the derive tremendous influence (Fig. 6 A) of polypeptide of their expressed Rubisco activating enzymes.Δ rca183H12 strain system is that every strain has more 50-100 silique than Δ rcaRCA1 strain, has more 40-80 silique than wild-type plant.In addition, the silique of Δ rca183H12 is bigger than wild-type plant and Δ rcaRCA1 strain system, and produces more seed (Fig. 6 B).26 ℃ down the silique of the Δ rca183H12 of growth to demonstrate the wild-type of growing under its phenotype and the normal growth condition similar, but the seed of generation is less.Observe under the normal growth condition, the Δ rca strain of expressing RCA1 and 183H12 is that its seed weight has decline (Fig. 6 C slightly than wild-type plant; The white rod).But, to observe and be exposed to continuously under 26 ℃, the seed weight of each strain system of Δ rca183H12 is big 50%-150% than each strain of wild-type plant or Δ rcaRCA1.With 26 ℃ down each strains system of growth seed weight with 22 ℃ down the seed weight of the same strain system of growth compare discovery, Δ rca183H12-2, Δ rca183H12-3 and Δ rca183H12-20 strain are the influence that obviously is not subjected to higher growth temperature than Δ rcaRCA1 strain system or wild-type.

Because being exposed to 26 ℃ causes producing little silique, contained seed amount is less, and seed weight is also little, so analyze the seed developmental potency with germination test.The seed of wild-type, Δ rcaRCA1-1 and the Δ rca183H12-3 plant that are collected in the normal growth condition and grow under 26 ℃ germinates down at 22 ℃ then.Seed in 22 ℃ of Δ rcaRCA1-1 that grow down and Δ rca183H12-3 parent plant system shows identical percentage of germination (86%), and this is than wild-type plant (94%) low slightly (Fig. 6 C).Observe the quilt that germinates from the seed of 26 ℃ of Δ rcaRCA1-1 parents collections of growing down and suppress (4%) fully, and remarkable suppress (26%) of wild type seeds quilt.On the contrary, the seed of collecting from 26 ℃ of Δ rca183H12-3 parents that grow down shows 70% higher relatively percentage of germination.

In addition, also analyzed photosynthesis speed, the speed of growth and the seed production of plant under following growth conditions.

Normal condition: plant is pressed illumination in 16 hours (225 μ mol photon m down at 22 ℃ ^-2s ^-1) and dark scheme growth in 8 hours.

The intensification condition: plant grew for two weeks under the normal growth condition, transferred to then in the growth case by illumination in 16 hours (225 μ mol photon m ^-2s ^-1) and dark scheme growth in 8 hours.In the illumination circulation, temperature is set to 22 ℃ and kept 6 hours, is rapidly heated then to 30 ℃ (2 ℃ of per minutes), keeps 4 hours.After the thermal treatment, temperature is set back 22 ℃.

Continuous intensification condition: plant grew for two weeks under the normal growth condition, transferred to then in the growth room by high illumination in 16 hours (300 μ mol photon m ^-2s ^-1) and dark scheme growth in 8 hours.In illumination/dark cycle, temperature is set at 26 ℃, suitably is set at 80%.

Below sum up the result that the Rubisco activating enzymes under above-mentioned each condition are derived and measured in the plant of polypeptide active.

The speed of growthUse chlorophyll fluorescence imaging system (FluorImager, Qubit SystemInc.) to measure the leaf area.Unconverted wild-type plant observed leaf area under different growth conditionss is set at 100%.Digital proof in the table 4 is expressed derive any plant of polypeptide of three kinds of Rubisco activating enzymes and is compared with the plant of the wild-type Rubisco activating enzymes of wild-type plant or express transgenic, and the speed of growth improves under the intensification condition.

Photosynthesis speed(LI6400 Li-Cor) analyzes the CO of each plant with portable infrared gas analyzer ₂Fixation case 15 minutes.Light source is set at 225 μ mol photon m ^-2s ^-1, by built-in CO ₂Injected system is fed to the CO of leaf ₂Level is 350 μ mol m ^-2s ^-1Digital proof in the table 5 is expressed derive any plant of polypeptide of three kinds of Rubisco activating enzymes and is compared with the plant of the wild-type Rubisco activating enzymes of wild-type plant or express transgenic, and photosynthesis speed improves (referring to the 3rd row) under the intensification condition.

Seed productionMeasure seed weight (mg) from sophisticated dry plant.Rate of emergence is measured by the quantity of the plant that germinates on the MS plate that replenishes with kantlex.Digital proof in the table 5 is expressed derive any plant of polypeptide of three kinds of Rubisco activating enzymes and is compared with the plant of the wild-type Rubisco activating enzymes of wild-type plant or express transgenic, and seed production improves (referring to the 4th row) under the intensification condition.Express derive any plant of polypeptide of three kinds of Rubisco activating enzymes and the raising (referring to table 6) of seed production under continuous intensification condition, also occurs.Express the derive plant of polypeptide 183H12 (SEQ ID NO:7) of Rubisco activating enzymes and compare with the plant of the wild-type Rubisco activating enzymes of wild-type plant or express transgenic, rate of emergence improves (referring to table 7) under intensification condition continuously.

Table 1: password sublist

Table 2: amino-acid substitution is to the influence of Rubisco activated enzyme activity and thermostability

^*The quantity of activation Rubisco under 40 ℃ accounts for the percentage ratio of the quantity of the activation Rubisco under 25 ℃.

The derive relative reactivity (%) of polypeptide of table 3:Rubisco activating enzymes

Table 4: the leaf area under the normal and intensification condition

^*The leaf area of the unconverted wild-type of Arabidopis thaliana is set at 100%.

Table 5: photosynthesis speed and seed production under the normal and intensification condition

The clone	Strain system numbering	Photosynthesis * (μmol CO 2m -2s -1)	Seed productive rate # (%)
				Wild-type	Unconverted	7.85±0.36	72±14
Wild-type (SEQ ID NO:1)	RCA1-1	7.25±1.36	74±12
				183H12 (SEQ ID NO：7)	183H12-3	9.41±0.66	87±8
382D8 (SEQ ID NO：15)	382D8-1	8.9±0.93	100±11
				301C7 (SEQ ID NO：19)	301C7-3	9.03±0.9	87±16

^*Monitor net photosynthesis at 30 ℃ after following 2 hours.

^#1000 seed weights of the plant that grows under the intensification condition account for the percentage ratio of 1000 seed weights of the plant that grows under the normal condition

Table 6: the seed production under the normal and continuous intensification condition

Table 7: the seed germination rate of the plant results of growing under the normal and continuous intensification condition

Sequence table

<110>Pioneer Hi-Bred International，Inc.

<120>RUBISCO ACTIVASE WITH INCREASEDTHERMOSTABILITY AND METHODS OF USE THEREOF

<130>V0136-PCT

<150>60/711,449

<151>2005-08-24

<160>26

<170>FastSEQ for Windows Version 4.0

<210>1

<211>1173

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400>1

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>2

<211>390

<212>PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400>2

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Met Ssr Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

Gln Leu ALa Glu Thr Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>3

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>3

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcagg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>4

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>4

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Arg Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>5

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>5

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg ataagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>6

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>6

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Ile Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>7

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>7

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga tttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca gacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctc tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>8

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>8

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Arg Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>9

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>9

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatggga 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>10

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>10

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Asn Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>11

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>11

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctacgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg ataagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgaaag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgctcgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>12

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>12

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Thr Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Ile Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Lau Gly Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>13

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>13

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctacgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga tttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtsa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca gacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgctcgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>14

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arsbidopsis thaliana) sequence

<400>14

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Thr Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Arg Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>15

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>15

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg gtgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga tttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtat atgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>16

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>16

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Val Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Ile Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Asn Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>17

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>17

atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cctcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtat atgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca gacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctc tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>18

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>18

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Leu Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Ile Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Arg Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Asn Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>19

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>19

atggtgaaag aagacaaaca aaccgacgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cctcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtat atgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa 960

ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgctcgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctt tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>20

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>20

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Leu Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Ile Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Asn Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>21

<211>1173

<212>DNA

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>21

atggtgaaag aagacaaaca aaccgacgga gacagatgga gaggtcttgc ctacgacact 60

tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt cttccaagct 120

cctatgggaa ccggaactca ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180

cttaggcagt acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg 240

gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat caaggttcca 300

cttattttgg gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc 360

atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga gagtggaaac 420

gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480

aagggaaaga tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt 540

ggtactactc agtacactgt caacaaccag atggttaacg caacactcat gaacattgct 600

gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc 660

cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat ccgtgatgga 720

cgtatggaga agttctactg ggccccgacc cgtgaagacc gtatcggtat atgcaagggt 780

atcttcagaa ctgacaagat caaggacgaa gacattgtca gacttgttga tcagttccct 840

ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900

aagttcgttg agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa 960

ggaccccccg tgttcgagca acccgagatg acttatgaga agcttatgga atacggaaac 1020

atgcttgtga tggaacaaga gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080

caggctgctc tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140

acagaggaaa aggagcccag caagctcgag taa 1173

<210>22

<211>390

<212>PRT

＜213〉artificial sequence

<220>

＜223〉based on Arabidopis thaliana (Arabidopsis thaliana) sequence

<400>22

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

1 5 10 15

Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly

20 25 30

Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His

35 40 45

Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr

50 55 60

Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met

65 70 75 80

Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn

85 90 95

Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly

100 105 110

Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro

115 120 125

Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro

130 135 140

Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys

145 150 155 160

Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala

165 170 175

Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val

180 185 190

Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu

195 200 205

Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys

210 215 220

Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly

225 230 235 240

Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly

245 250 255

Ile Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile

260 265 270

Val Arg Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly

275 280 285

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

290 295 300

Ser Leu Gly Val Glu Asn Ile Gly Lys Arg Leu Val Asn Ser Arg Glu

305 310 315 320

Gly pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met

325 330 335

Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val

340 345 350

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

355 360 365

Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys

370 375 380

Glu Pro Ser Lys Leu Glu

385 390

<210>23

<211>17

<212>DNA

＜213〉artificial sequence

<220>

<223>primer

<400>23

cagacaatgt tggcctc 17

<210>24

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<223>primer

<400>24

acgagtaacg atggtagg 18

<210>25

<211>15

<212>DNA

＜213〉artificial sequence

<220>

<223>primer

<400>25

gtctatacct tgagc 15

<210>26

<211>14

<212>DNA

＜213〉artificial sequence

<220>

<223>primer

<400>26

tcagtcatac tcgg 14

Claims (20)

1. isolated nucleic acid molecule, described nucleic acid molecule comprise any one or its complementary sequence among the SEQ ID NO:3,5,7,9,11,13,15,17,19,21.

2. isolated nucleic acid molecule, described nucleic acid molecule is selected from:

A. the nucleic acid molecule that comprises such nucleotide sequence, any one among this nucleotide sequence and the SEQID NO:3,5,7,9,11,13,15,17,19,21 or the nucleotides sequence of its complementary sequence are shown at least 95% identity;

B. the encode nucleic acid molecule of such polypeptide, described polypeptide comprises any one aminoacid sequence among the SEQ ID NO:4,6,8,10,12,14,16,18,20,22;

C. under stringent condition with the nucleic acid molecule of such nucleic acid probe hybridization, described nucleic acid probe is made up of any one or the nucleotide sequence of its complementary sequence among the SEQ ID NO:3,5,7,9,11,13,15,17,19,21.

3. claim 1 or 2 isolated nucleic acid molecule, wherein said nucleic acid molecule encoding is compared the polypeptide that thermostability improves with the polypeptide of SEQ ID NO:2.

4. carrier, described carrier comprises the nucleic acid molecule of claim 1 or 2.

5. the carrier of claim 4, described carrier is an expression vector.

6. host cell, described host cell comprises the carrier of claim 4.

7. isolated polypeptide, described polypeptide comprises any one among the SEQ ID NO:4,6,8,10,12,14,16,18,20,22.

8. isolated polypeptide, described polypeptide is selected from:

A. with SEQ ID NO:4,6,8,10,12,14,16,18,20,22 in any one aminoacid sequence the polypeptide of at least 95% identity is arranged;

B. by the polypeptide of such nucleic acid molecule encoding, described nucleic acid molecule comprise with SEQID NO:3,5,7,9,11,13,15,17,19,21 in any one or its complementary sequence nucleotide sequence that at least 95% identity is arranged;

C. by the polypeptide of such nucleic acid molecule encoding, described nucleic acid molecule under stringent condition with the nucleic acid probe hybridization of forming by the nucleotide sequence of any one or its complementary sequence among the SEQ ID NO:3,5,7,9,11,13,15,17,19,21.

9. claim 7 or 8 isolated polypeptide, wherein said polypeptide is compared thermostability and is improved with the polypeptide of SEQ ID NO:2.

10. transgenic plant, the transgene expression that described transgenic plant comprise:

Any one polypeptide among the a.SEQ ID NO:4,6,8,10,12,14,16,18,20,22; Or

Any one nucleic acid molecule among the b.SEQ ID NO:3,5,7,9,11,13,15,17,19,21.

11. the transgenic plant of claim 10, wherein said plant are selected from corn, tomato, potato, paddy rice, soybean, cotton, Sunflower Receptacle, clover, lettuce, canola oil dish, Chinese sorghum or tobacco plant.

12. comparing thermotolerance with non-transgenic plant, the transgenic plant of claim 11, wherein said transgenic plant improve.

13. transgenic plant, the transgene expression that described transgenic plant comprise:

C. the polypeptide of claim 8, or

D. the nucleic acid molecule of claim 2.

14. the transgenic plant of claim 13, wherein said plant are selected from corn, tomato, potato, paddy rice, soybean, cotton, Sunflower Receptacle, clover, lettuce, canola oil dish, Chinese sorghum or tobacco plant.

15. comparing thermotolerance with non-transgenic plant, the transgenic plant of claim 13, wherein said transgenic plant improve.

16. a stable on heating method that improves plant, described method are included in any one polypeptide of expressing in this plant among the SEQ ID NO:4,6,8,10,12,14,16,18,20,22.

17. the method for claim 16, wherein said polypeptide is expressed in one or more plastids of this plant.

Photosynthesis speed improves under the intensification condition 18. the method for claim 16, wherein said polypeptide expression cause.

19. the transgenic plant of claim 13, the endogenous ribulose-1,5-bisphosphate of wherein said plant, the expression of 5-bisphosphate carboxylase/oxygenase activating enzymes or activity are regulated downwards.

20. the transgenic plant of claim 13, the endogenous ribulose-1,5-bisphosphate of wherein said plant, the expression or the activity of 5-bisphosphate carboxylase/oxygenase activating enzymes are eliminated.

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