CN101365786A

CN101365786A - Plants having improved growth characteristics and methods for making the same

Info

Publication number: CN101365786A
Application number: CNA2006800521587A
Authority: CN
Inventors: V·弗兰卡德; C·勒佐; A·I·桑兹莫林纳罗; C·达曼
Original assignee: CropDesign NV
Current assignee: CropDesign NV
Priority date: 2005-12-01
Filing date: 2006-11-29
Publication date: 2009-02-11
Anticipated expiration: 2026-11-29
Also published as: CN101365786B; ZA200805634B

Abstract

The present invention relates generally to the field of molecular biology and concerns a method for improving various plant growth characteristics by modulating expression in a plant of a nucleic acid encoding a GRP (Growth-Related Protein). The present invention also concerns plants having modulated expression of a nucleic acid encoding a GRP, which plants have improved growth characteristics relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention. The GRP may be one of the following: Seed Yield Regulator (SYR), FG-GAP1 CYP90B, CDC27, AT-hook transcription factors, DOF transcription factors and Cyclin Dependent Kinase Inhibitors (CKIs).

Description

Plant and production method thereof with growth characteristics of improvement

Technical field

Relate generally to biology field of the present invention and relate to the method that improves the various plants growth characteristics by the expression of nucleic acids of in plant, regulating coding GRP (growth associated protein).The invention still further relates to the plant of the GRP coding nucleic acid expression with adjusting, this plant is compared the growth characteristics with improvement with corresponding wild type plant or other control plants.The present invention also provides the construct that is used for method of the present invention.

Background technology

In view of world population constantly increases, the obtainable land area that is used for agricultural constantly reduces, and the diversity that improves farm efficiency and increase gardening plant remains the major objective of research.The ordinary method utilization that is used for the improvement of farm crop and gardening plant selects breeding technique to identify the plant with desired characteristic.Yet, this type of selects breeding technique to have several shortcomings, be that these technology are normally labor-intensive and cause producing the plant that comprises heterogeneous hereditary fill-in usually, described heterogeneous hereditary fill-in may always not cause transmitting from mother plant the proterties of expectation.Molecular biological progress has made the mankind can operate the idioplasm (germplasm) of animal and plant.The genetically engineered of plant need be separated and operate genetic material (form with DNA or RNA exists usually) and subsequently genetic material be imported plant.Such technology has caused producing the plant of economy, agronomy or gardening proterties with various improvement.Proterties with special economic implications is a for example high yield of growth characteristics.Output is normally defined the production of measurable tool economic worth from farm crop.This can define aspect quantity and/or the quality.Output directly depends on Several Factors, for example the number of organ and size, plant structure (for example, ramose number), seed production etc.The growth of root, dietetic alimentation and stress tolerance/resistance (stresstolerance/resistance) also can be the important factors of determine output.

Seed production is the proterties of particularly important, because the seed of many plants is very important for the nutrition of humans and animals.No matter be consumption by direct seed itself or the consumption by the meat product on the seed that is based upon processing, farm crop for example corn, rice, wheat, canola and soybean have occupied over half that total people's heat takes in.They are the source of the meta-bolites that is used for industrial processes of sugar, oil and numerous species still.Seed comprises embryo (the new bud and the source of root) and endosperm (source of nutrition that is used for embryonic development in the early growth of sprouting and seedling).The growth of seed involves many genes, and meta-bolites need be transferred to the seed of growth from root, leaf and stem.Endosperm can assimilate carbohydrate, oil and proteinic metabolic precursor thereof especially, and it is synthesized storage macromole filling seed.

Another important character of many farm crop is early growth gesture (early vigour).Improving the early growth gesture in temperate zone and tropical rice growing kind is the free-revving engine of the modern rice procedure of breeding.It is very important that long root for the suitable soil anchor of water direct seeding rice.When directly flood field (flooded field) is gone in sowing with rice and when plant must be emerged the water surface fast, longer stem will be related with growth potential.When carrying out bar sowing time, longer mesocotyl and coleoptile are particularly importants for good coming up.The early growth gesture also can be caused by the plant adaptability that increases, and the increase of described plant adaptability can be owing to plant for example to the better adaptation (promptly more can tackle the various abiotic or biological factors of coercing) of its environment.Plant with early growth gesture also shows the neat seedling of better crop (establishment of the crop), and (farm crop grow in the mode of homogeneous more, be most of plant reaches growth in the substantially the same time each stage) and show better growth and common higher output.

An important character is the abiotic stress patience that improves again.Abiotic stress is the major cause of world wide crop loss, and it makes the mean yield of most of staple crops plant subtract (people such as Wang, Planta (2003) 218:1-14) more than 50%.Abiotic stress can be caused by arid, salinity, extreme temperature, chemical toxicity and oxidative stress.For worldwide peasant, the ability that improves plant tolerance abiotic stress has huge economic advantages, and it makes can be under disadvantageous condition and regional culture farm crop that originally may not crop culture.

Therefore can increase crop yield by a factor in the above-mentioned factor of optimization.

Depend on end-use, the improvement of some yield traits may be better than the improvement of other proterties.For example for such as application such as feed or timber production or biofuel sources, may expect to increase the leaf part of plant, for such as application such as flour, starch or oil production, the increase of planting subparameter may be to expect especially.Even in kind of subparameter, depending on purposes, some parameters can be better than other parameters.Different mechanism can be facilitated the seed production of increase---and no matter its form for the seed size of increase still is the form of the number seeds of increase.

A method that increases (seed) output in plant can be the intrinsic growth mechanism that changes plant.Such mechanism is the cell cycle.

Have now found that and to improve the various growth characteristics of plant by the expression of nucleic acid in plant of regulating the GRP (growth associated protein) in the coded plant.GRP can be one of following: seed production conditioning agent (Seed Yield Regulator) (SYR), FG-GAP, CYP90B, CDC27, AT hook transcription factor (AT-hook transcription factars), DOF transcription factor and cell cycle protein dependent kinase inhibitor (CKI).

Background

Seed production conditioning agent (SYR)

Existence increases the lasting needs of gene to finding new seed production, used so far for example except that other strategies, by controlling hormone levels (WO 03/050287), by manipulation cell cycle (WO 2005/061702), by controlling the several method that the gene (WO2004/058980) that participates in the salt stress reaction carries out.

SYR is the new albumen that is not characterized yet so far.SYR shows certain homology (sequence identity of about 48% on dna level to Arabidopsis plant (Arabidopsis) albumen that is called ARGOS, about 45% sequence identity on protein level) (people such as Hu, Plant Cell15,1951-1961,2003; US 2005/0108793).People such as Hu infer albumen that ARGOS is the tool unique function and are encoded by individual gene.To cross the main phenotype of expression be blooming of the leaf biomass that increases and delay to ARGOS in the Arabidopsis.

FG-GAP

FG-GAP albumen is the transmembrane protein of supposition.It is characterized in that the existence of the membrane spaning domain in existence of one or more FG-GAP structural domain (Pfam accession number PF01839) and proteinic N-terminal signal peptide and C-terminal half part.

Such protein D EX1 separates from the Arabidopsis plant and work during pollen development according to reports people Plant Physiol.127 such as (, 1739-1749,2001) Paxson-Sowders.The Dex1 mutant plant is presented in the pollen wall pattern formation (pattern formation) has defective.The DEX1 genes encoding is positioned to 896 amino acid whose albumen of plasma membrane through prediction, and wherein residue 1 to 860 is positioned at the cell outside, and residue 880 to 895 is positioned at the tenuigenin side of film, and residue 861 to 879 is the potential membrane spaning domain.12 potential N glycosylation sites are present among the DEX1.Therefore, this protein have modified by severe and with the various ingredients of cell walls is interactional may.DEX1 shows from the proteic maximal sequence similarity of the hemolysin sample of vibrio cholerae (V.cholerae), yet about 200 amino acid whose fragments (amino acid 439-643) of DEX1 also show the limited similarity to the calcium binding domains of alpha-6 integrin.Have the calcium binding partner of at least 2 groups supposition in this zone, it also is present in the Arabidopsis calmodulin (AC009853) of prediction.Therefore, as if DEX1 may be a calcium binding protein.The vegetable-protein that DEX1 is seemingly unique; In bacterium, fungi or animal, there is not homologue.

Formed several suppositions (people such as Paxson-Sowders, 2001) by the structure of the DEX1 of observed change and prediction in the dex1 plant about the effect of this albumen in pollen wall forms:

(a) DEX1 may be a linker albumen.It can combine with the sporule film and participate in prototheca (primexine) or sporopollenin (sporopollenin) are attached to plasma membrane.The sporule surface lacks the structural modification that this albumen can cause prototheca.Many potential N glycosylation sites were consistent with DEX1 and callose wall, intine or both adhering to.

(b) DEX1 may be the component of prototheca matrix and may work in the initial polymerization of prototheca.Ca ^{+ 2}As if the change of ionic concn synthetic extremely important to pollen wall; The beta-glucan synthase is subjected to the Ca of micro-molar concentration in callose wall forming process ^{+ 2}Activation.

(c) DEX1 may be the part of asperities ER and the processing that participates in the prototheca precursor and/or to the transportation of film.Conforming to the overall shortage of prototheca precursor with overall the change appears in the delay of prototheca.Prototheca matrix is made up of polysaccharide, protein and Mierocrystalline cellulose at first, incorporates the material that has more resistance then into.Therefore, DEX1 may participate in the formation or the transportation of many different componentss.

CYP90B

The plain steroid of rape (BR) is that a class is for promoting plant-growth, division and the very important plant hormone of growth.Term BR totally is meant the polyhydroxylated steroid derivatives of the natural generation of kind more than 40, and it has the structural similarity with the animal steroid hormone.In these plant hormones, brassinolide shows tool biologic activity (about summary, Clouse (2002) Brassinosteroids.TheArabidopsis Book:1-23).

Used biological chemistry and mutation analysis to illustrate the BR biosynthetic pathway.BR is by at least two branch's bio-chemical pathways that start from identical initial precursor campesterol synthetic people (1997) Physiol Plant 100:710-715 such as () Fujioka.Found the most of Codocyte pigment of the BR biosynthesis gene of being found P450 monooxygenases (CYP) (Bishop and Yokota (2001) Plant CellPhysiol 42:114-120).The oxidation of the many chemical substances of CYP enzyme superfamily catalysis, herein, the essential oxidizing reaction in the catalysis BR biosynthesizing more specifically.Intermediate product campestanol that a step in the important step of having identified is hydroxylation BR and the steroid side chain of 6-oxo campestanol, thus the 6-deoxidation formed respectively for cathasterone and cathasterone.These two parallel oxidation steps always are also referred to as early stage steroid C-22 α-hydroxylation step (people (1998) Plant Cell 10:231-243 such as Choe).In Arabidopsis, specific CYP enzyme CYP90B1 or DWF4 carry out this step (the general reference about plant CYP nomenclature is seen, people such as Nelson (2004) PlantPhys 135:756-772).

Lack the arabidopsis mutant type plant of steroid 22 α hydroxylase activities owing to inserting T-DNA in the DWF4 locus, show owing to lacking the dwarfing phenotype that cell elongation causes people (1998) Plant Cell 10:231-243 such as () Choe.The biological chemistry of using BR biosynthesizing intermediate product to carry out is fed and be studies show that all downstream compounds have all been saved this phenotype, and known precursor then can not.

Use the cauliflower mosaic virus 35S promoter to produce dystopy and cross the transgenic arabidopsis of expressing Arabidopis thaliana DWF4 genomic fragment and tobacco plant (both is dicotyledons) (people (2001) Plant J 26 (6) such as Choe: 573-582).The sum of plant height, branched sum and seed when the phenotypic characteristic of plant shows hypocotyl length, maturation is compared with control plant, obtains increasing in transgenic plant.People such as Choe find that the seed production that increases has the more seed of more number owing to every strain plant, in the scope that is increased in standard deviation of seed size.In WO00/47715, further describe these experiments.

Patent US 6,545, and 200 relate to the nucleic acid fragment of coding sterol biosynthesis gene, and more specifically claimed coding has C-8, the nucleotide sequence of the polypeptide of 7 sterol isomerase activities.The partial nucleotide sequence of encoding D WF4 is disclosed.

US 2004/0060079 relates to generation and has the monocotyledonous method of improvement of anticipant character.Provide the nucleotide sequence (being called OsDWF4 or CYP90B2) of the rice DWF4 that wherein will encode to place example under constitutive promoter (rice actin promoter) control.There are 14 strains to show in the transgenosis rice of 36 strains expression chimeric construct body, compare every fringe Number of kernels of increase with non-conversion control plant.According to the contriver, the output increase of comparing transgenic plant with wild-type is because the overall number increase of seed causes, because do not find significant difference on " 10 weights ".

CDC27

Depend on final purposes, the improvement of some yield traits can be better than other proterties.For example for application examples such as feed or timber production, or the biofuel source, the increase of leaf part may expect that for the production of application examples such as flour, starch or oil, the increase of planting subparameter may be to expect especially.Even in kind of subparameter, depending on purposes, some parameters can be better than other parameters.Different mechanism can promote to increase seed production---no matter its with form of the seed size that increases still with the form of the number seeds that increases.Such mechanism is the cell cycle.

By the cell cycle develop g and D for all multicellular organisms be essence and be vital for cell proliferation.The main ingredient of cell cycle is a high conservative in yeast, Mammals and plant.Cell cycle is divided into following period in succession: G0-G1-S-G2-M usually.Dna replication dna or synthetic usually in (DNA is synthetic in " S " expression) generation of S phase, chromosomal mitotic division is separated in M phase (" M " represents mitotic division) generation, be inserted with interval (gap phase) therebetween, G1 (dna replication dna before cell vegetative period) and G2 (behind the dna replication dna, cell preparation splitted period).After division of cytoplasm (final step of M phase), finish cell fission.Withdrawed from the cell cycle and become the immobilized cell and be regarded as being in the G0 phase.Can stimulate the cell that is in this phase to reenter the cell cycle in the G1 phase." G " representative " interval " among G1, G2 and the G0.Cell cycle progression finish the complete copy that makes daughter cell in fission process, can accept the parental gene group.

Cell fission is subjected to two main cell cycle events, and promptly DNA synthetic initial sum is mitotic initial, control.The conversion of each incident in these critical events each time all is subjected to the control as the specified protein mixture of restriction point (checkpoint) (participating in dna replication dna and division).In Mammals and vegetable cell, the synthetic necessary expression of gene of DNA is subjected to regulation and control (La Thangue, 1994 of transcription factor E2F family on the G1/S border; People such as Muller, 2001; People such as De Veylder, 2002).Cell cycle enter by the E2F/Rb mixture to regulate and control/trigger, described E2F/Rb mixture integrated signal also makes it possible to transcribing of activating cells cycle genes.Conversion between the different times of cell cycle, and the development of passing through the cell cycle thus, forming and activating and drive by different different dimerization serine/threonine protein kitase (being commonly referred to cell cycle protein dependent kinase (CDK)).These kinase whose active preconditions are and the physical bond of specific cells cyclin, activate the expression that the time of taking place depends primarily on cyclin.The combination of cyclin will be induced the N of bonded CDK to hold the conformational change of leaf and be promoted the location and the substrate specificity of mixture.It is activated when monomer CDK combines with cyclin, thereby has kinase activity.The level of cyclin fluctuateed in the cell cycle, from but determine that CDK activates the principal element of time of origin.In the cell cycle process, the periodicity that comprises these mixtures of cyclin and CDK activates the time that has mediated cell cycle conversion (restriction point) and regulates.

Exist and guarantee that mechanism once only takes place dna replication dna in the cell cycle.For example, CDC16, CDC23 and CDC27 albumen be called anaphase-promoting complex (anaphase promotingcomplex) (APC) or the part of the high molecular weight component of cell cycle body (referring to Romanowski and Madine, Trends in Cell Biology 6,184-188,1996, with Wuarin and Nurse, Cell 85,785-787 (1996).This mixture in the yeast is made up of at least 8 albumen, PROTEIN C DC16, CDC23 and CDC27 and 5 other subunits (people 1996 such as Peters that are called APC1, APC2, APC4, APC5 and APC7 of comprising TPR-(three tetradecapeptide repeating units), Science274,1199-1201).Its substrate of APC target is by catalysis ubiqutin molecule and being connected of these substrates carrying out proteolytic degradation.The hydrolysis of APC dependence protein matter be mid-term sister strand when the later stage changes separate and finally withdraw from necessary from mitotic division.Later stage inhibitor protein Pds1p and mitotic cell cyclin for example cell periodic protein B be APC substrate (people 1998 such as Ciosk, Cell 93,1067-1076; People such as Cohen-Fix 1996, Genes Dev 10,3081-3093; People such as Sudakin 1995, Mol Biol Cell 6,185-198; People such as Jorgensen 1998, Mol Cell Biol 18,468-476; Townsley and Ruderman 1998, Trends CellBiol 8,238-244).In order to produce the activity of ubiqutin ligase enzyme, at least CDC16, CDC23 and CDC27 need the M phase by phosphorylation (Ollendorf and Donoghue 1997, J Biol Chem272,32011-32018).Activated APC continued to exist in the whole G1 phase of subsequently cell cycle, thereby prevent the too early appearance of type B cell cyclin, described too early appearance can cause entering the S phase uncontrollably (Irniger and Nasmyth 1997, J Cell Sci 110,1523-1531).In the middle of the sudden change of any all can cause among at least 2 the component CDC16 of proof APC and the CDC27 in yeast the DNA over-replicate by the M phase (Heichman and Roberts 1996, Cell 85,39-48).Mitotic division and fissional this nuclear dna replication dna process of not having subsequently are called the DNA endoreduplication, and it causes the cell size that increases.

CDC16, CDC23 and CDC27 comprise the 34 (TPR of peptide repeating unit; 34 amino acid longs) albumen.The minimum consensus sequence of the suggestion of TPR primitive (motif) is as follows: X ₃-W-X ₂-L-G-X ₂-Y-X ₈-A-X ₃-F-X ₂-A-X ₄-P-X ₂, wherein X be any amino acid (people 1994 such as Lamb, EMBO J 13,4321-4328).Consensus sequence can be showed significant degeneracy, and has minimum homology or do not have homology in non-total residue.The hydrophobicity of important total residue seemingly and size but not its identity.In yeast and higher eucaryote, in mitotic division (comprising AC protein ingredient CDC16, CDC23 and CDC27), transcribe, the input of montage, protein and neural (Goebl and the Yanagida 1991 of taking place, Trends Biochem Sci 16, all there is the TPR primitive therein in the multiple protein that works in 173-177).TPR forms αLuo Xuanjiegou; Series connection repeating unit be organized into the superhelix that is ideally suited as the protein identification interface (Groves and Barford1999, Curr Opin Struct Biol 9,383-389).In the α spiral, there are 2 amphipathic structural domains usually, one is positioned at NH ₂End regions, (people 1990 such as Sikorski, Cell 60,307-317) near the COOH end regions for another.

From different bioseparation CDC27 (be also referred to as Hobbit; Other titles comprise CDC27, BimA, Nuc2 or makos), described biology comprises Aspergillus nidulans (Aspergillus nidulans), yeast, fruit bat, people and each kind of plant (for example Arabidopis thaliana (Arabidopsis thaliana) and rice (Oryzasativa)).The gene of coding CDC27 is present in most of genomes with single copy, but can for example find 2 copies in the Arabidopis thaliana in identical genome exceptionally.Proteic these 2 genes of coding CDC27 are called after CDC27A and CDC27B (being respectively MIPS call number At3g16320 and At2g20000).

Disclosed International Patent Application WO 01/02430 has been described CDC27A (CDC27A1 and CDC27A2) and CDC27B sequence.Also in the document, described wherein from NH ₂End regions has lacked the CDC27B aminoacid sequence of 161 amino acid whose brachymemmas.About being coded in NH ₂The CDC27B gene of the CDC27B polypeptide of end regions brachymemma has been quoted GenBank accession number AC006081 in the document.Reported in literature NH ₂End regions is guarded in the CDC27 of different sources homologue.The CDC27 sequence of mentioning among the WO01/02430 is described to can be used for changing endoreduplication.

The DNA endoreduplication betides in the flowering plant natively, for example during seed development.The DNA endoreduplication causes having the nucleus of expansion of the dna content of raising.Someone thinks that the dna content that increases during the endoreduplication may provide the genetic expression of increase between endosperm development and seed influx time, because it conforms to protein accumulation with enzymic activity that this time go up to increase people such as (, (1992) Genet.Eng.14:65-88) Kowles.In the cereal species, the cell endosperm by the endoreduplication sign period stored seeds reserve.The magnitude of DNA endoreduplication and endosperm fresh weight height correlation, this is hinting the vital role of DNA endoreduplication in determining endosperm quality people (2000) Plant Cell Environ.23:657-663 such as () Engelen-Eigles.In corn for example, endosperm accounts for 70 to 90% of seed quality (kernel mass); Therefore, mediate the factor of endosperm development, also can determine the seed production of corn by the heavy mode of simple grain to a great extent.Thereby the endoreduplication of increase is indicated the seed biomass of increase usually, but never relevant with the number seeds that increases.

AT hook transcription factor

In belonging to the polypeptide of the transcription factor family relevant, there is AT hook structure territory with Chromatin Remodeling (Chromatin remodeling).AT hook primitive is made up of about 13 (about sometimes 9) amino acid, these amino acid participate in DNA in conjunction with and have a preference to the zone of being rich in A/T.In Arabidopis thaliana, there are at least 34 albumen that comprise AT hook structure territory.These albumen are enjoyed homology on the major part of sequence, wherein AT hook structure territory is special high conservative region.

International Patent Application WO 2005/030966 has been described the purposes of plant that the plant transcription factor in the several AT of comprising hook structures territory and this type of transcription factor are used to produce the stress tolerance of biomass with increase and increase.This application relates to the member of transcription factor G1073 clade, and point out: " use of tissue specificity or inducible promoter can relax to cross with G1073 clade member's composing type and express the relevant morphology effect of not expecting (for example, when the size of increase be when not expecting) ".The data that provide in this application relate to dicotyledons.

Opposite with these instructions, have now found that no matter to express and drive or drive in the tissue specificity mode by constitutive promoter, the expression of polynucleotide in monocotyledons that coding comprises the AT hook transcription factor (it comprises the member of clade G1073) of DUF296 structural domain all causes, compare with suitable control plant, do not have the plant that biomass increases hardly or fully.This shows that the relevant instruction of expressing this type of transcription factor in dicotyledons may not so easily be used for monocotyledons.Find also that now any of seed production who obtains is increased in degree or depends on employed tissue-specific promoter in nature.

The DOF transcription factor

The Dof domain protein is that the DNA binding domains with high conservative (has single C ₂-C ₂Zinc refers to) the plant idiosyncratic transcription factor.In the past ten years, in unifacial leaf and dicotyledons (comprising corn, barley, wheat, rice, tobacco, Arabidopis thaliana, summer squash, potato and pea), many Dof domain proteins have been identified.Shown that the Dof domain protein is used as transcriptional activator in various plants specific biological process or thing is met in resistance.

Cell cycle protein dependent kinase inhibitor (CKI)

The ability that increases plant seed output (no matter being) by number seeds, seed biomass, seed development, seed plumpness or any other seed correlated character agricultural and even during the biotechnology of many non-agricultural application examples such as material (for example medicine, antibody or vaccine) produces, all have many purposes.The change that increases the intrinsic growth mechanism that a method of seed production in the plant can be by plant realizes.

The intrinsic growth mechanism of plant is to be referred to as the continuous incident of the high-sequential of ' cell cycle '.By cell cycle development be all multicellular organisms g and D the basis and be vital for the propagation of cell.The main ingredient of cell cycle is a high conservative in yeast, Mammals and plant.Cell cycle is divided into following period in succession: G0-G1-S-G2-M usually.Dna replication dna or synthetic usually in (DNA is synthetic in " S " expression) generation of S phase, chromosomal mitotic division is separated in M phase (" M " represents mitotic division) generation, its interbody spacer is with interval, G1 (the cell vegetative period before the dna replication dna) and G2 (behind the dna replication dna cell preparation splitted period).After division of cytoplasm (final step of M phase), finish cell fission.Withdraw from the cell cycle and become the immobilized cell and be regarded as being in the G0 phase.Can stimulate the cell that is in this phase to make it reentering the cell cycle in the G1 phase." G " representative " interval " among G1, G2 and the G0.The cell cycle process finish the complete copy that makes each daughter cell can between cell division phase, accept the parental gene group.

Cell fission is subjected to two main cell cycle events, and promptly DNA synthetic initial sum is mitotic initial, control.Controlling each conversion of each incident in these critical events by specified protein mixture (participating in dna replication dna and the division) restriction point (checkpoint) that constitutes.In Mammals and vegetable cell, the synthetic necessary expression of gene of DNA is subjected to regulation and control (La Thangue, 1994 of transcription factor E2F family on the G1/S border; People such as Muller, 2001; People such as DeVeylder, 2002).Cell cycle enter by the E2F/Rb mixture to regulate and control/trigger, described E2F/Rb mixture integrated signal also makes transcribing of cell cycle gene to be activated.Conversion between the different times of cell cycle, and the progress by the cell cycle thus, forming and activating and drive by different different dimerization serine/threonine protein kitase (being commonly referred to cell cycle protein dependent kinase (CDK)).These kinase whose active preconditions are and the physical bond of specific cells cyclin that the activated time of origin depends primarily on the expression of cyclin.The combination of cyclin will be induced the conformational change of the N petiolarea of bonded CDK, thereby facilitate the location and the substrate specificity of mixture.It is activated when monomer CDK combines with cyclin, thereby has kinase activity.The level of cyclin fluctuateed in the cell cycle, from but determine the principal element of CDK activated time of origin.The periodicity that comprises these mixtures of cyclin and CDK in the cell cycle process activates the time that has mediated cell cycle conversion (restriction point) and regulates.Regulation and control CDK active other factors comprise that cell cycle protein dependent kinase inhibitor (CKI or ICK, KIP, CIP, INK), CDK activate kinases (CAK), CDK Phosphoric acid esterase (Cdc25) and CDK subunit (CKS) (people 1999 such as Mironov; Reed 1996).

According to the experiment of the endosperm that uses corn seed infer the inhibitor that has mitotic division CDK (Grafi and Larkins (1995) Science 269,1262-1264).After this, at different plant species Arabidopis thaliana (people (1997) Nature 386 (6624): 451-2 such as Wang for example; People (2001) Plant Cell 13:1653-1668 such as De Veylder; People such as Lui (2000) Plant J 21:379-385), tobacco (people (2002) Plant Physiol 2,002 130 (4) such as Jasinski: 871-82), identified several CKI in red autumnal leaves lamb's-quarters (Chenopodium rubrum) (people (1999) Plant Phys 120:339 such as Fountain) or the corn (corn) (people (2005) Plant Physiol 138:2323-2336 such as Coelho).Encoded protein is characterised in that, shows and p21 ^Cip1/ p27 ^Kip1/ p57 ^Kip2Aminoterminal cyclin/Cdk binding domains of the animal CKI of type has about 45 carboxyl terminal amino acid fragments of homology.Outside this carboxyl terminal zone, plant CKI shows extremely low homology.

Describe coding in Monsanto Technology LLC disclosed International Patent Application WO 2005/007829 under one's name and had the multiple isolated nucleic acid molecule of the active polypeptide of cell cycle protein dependent kinase inhibitor.

Disclosed International Patent Application WO 02/28893 and WO 99/14331 (both at CropDesign N.V. under one's name) have described different plant cell cycle protein dependent kinase inhibitors.In these applications, mentioned that these inhibitor are used to increase the purposes of output.

Summary of the invention

Now find in plant to increase the plant that proteic activity of SYR and/or the proteic expression of nucleic acids of coding SYR will cause comparing with corresponding wild type plant the growth velocity of seed production with increase and/or increase surprisingly.Now also find surprisingly, SYR crossing in rice expressed the main seed production that increases, (the main phenotype of expressing with the mistake of ARGOS in Arabidopis thaliana is opposite and leaf biomass and flowering time are not subjected to tangible influence, described main phenotype shows the blooming of the leaf biomass that increases and delay (people such as Hu, Plant Cell 15,1951-1961,2003; US2005/0108793)).

According to one embodiment of the invention, provide to be used to increase the seed production of plant and/or the method for growth velocity, this method is included in to be increased the active of SYR polypeptide or its homologue and/or increases this type of proteic expression of nucleic acids of coding in the plant; Randomly selection has the plant of the growth characteristics of improvement.

Advantageously, in relating to the scope of SYR, implement method of the present invention and cause producing such plant, described plant has the growth characteristics of multiple improvement, for example the seed production of Ti Gaoing and do not influence the biomass (when comparing with corresponding control plant) of trophicity plant part and life cycle suitable with corresponding control plant and do not have the delay of flowering time.More advantageously, the enforcement of the inventive method causes having the plant to the patience of abiotic stress of comparing raising with corresponding wild type (or other contrasts) plant.

Now find in plant to regulate proteic activity of FG-GAP and/or the proteic expression of nucleic acids of coding FG-GAP surprisingly and can cause producing with corresponding wild type plant and compare growth characteristics, particularly the plant of the output of Zeng Jiaing with improvement.

According to another embodiment of the invention, the method of the growth characteristics that is used to improve plant is provided, and it is included in regulates the active of FG-GAP polypeptide or its homologue and/or regulates the expression of nucleic acids of coding FG-GAP polypeptide or its homologue and plant that randomly selection has the growth characteristics of improvement in the plant.

Advantageously, in the scope that relates to FG-GAP polypeptide or its homologue, the enforcement of the inventive method causes having the growth characteristics of multiple improvement, the plant of for example cell fission of the structure of the biomass of the output of the growth of Ti Gaoing, raising, raising, improvement or raising (each all with corresponding wild type plant Comparatively speaking).Preferably, the growth characteristics of improvement comprises the output of comparing increase with corresponding wild type plant at least.

Find surprisingly that now the non-constitutive expression that increases the nucleic acid of coding CYP90B polypeptide or its homologue in plant can produce the plant of comparing the output with increase with suitable control plant.

According to an embodiment more of the present invention, the method that is used to increase plant biomass is provided, this method comprises the non-constitutive expression of nucleic acid in plant that increases coding CYP90B polypeptide or its homologue.

Have now found that the expression of nucleic acids that preferentially increases coding CDC27 polypeptide in axis end meristematic tissue can produce the plant of comparing the number seeds with increase with suitable control plant, described CDC27 polypeptide is at the NH of polypeptide ₂End regions has the TPR structural domain of at least one inactivation.

Therefore the invention provides the method for the number seeds (comparing with the number seeds of the control plant that suits) that is used to increase plant, this method comprises preferentially increases the NH that is coded in polypeptide in axis end meristematic tissue ₂End regions has the expression of nucleic acids of CDC27 polypeptide of the TPR structural domain of at least one inactivation.

Have now found that the expression of nucleic acids that preferentially increasing encodes comprises the AT hook structure territory and the polypeptide of DUF296 structural domain can produce with suitable control plant to be compared, and has the plant of the seed production of increase in monocotyledonous endosperm tissue.

Therefore an embodiment more of the present invention provides and has been used for comparing the method that increases monocotyledonous seed production with suitable control plant, and this method comprises preferentially increases the expression of nucleic acids that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain in monocotyledonous endosperm tissue.

Have now found that the expression of nucleic acid in plant that increases encoding D OF transcription factor polypeptide can produce the plant of comparing the output with increase with suitable control plant.

According to an embodiment more of the present invention, the method that is used to increase plant biomass is provided, this method comprises the expression of nucleic acid in plant that increases encoding D OF transcription factor polypeptide.

Have now found that the preferential expression decreased of endogenous CKI gene in the albumen tissue can produce, the seed production that the preferential plant that reduces is not taken place with the expression of endogenous CKI gene in the albumen tissue is wherein compared, and has the plant of higher seed production.Thereby the invention provides the method that is used for comparing the seed production that increases plant with suitable control plant, this method comprises the preferentially endogenous CKI expression of gene of minimizing in the endosperm tissue of plant.

Detailed Description Of The Invention

Ding Yi term " output of increase " is meant with corresponding wild type or other control plants and compares herein, the increase of the biomass (weight) of one or more parts of plant (particularly can gather in the crops part), the increase of this biomass can be the ground or subterranean.The increase of underground biomass is attributable to for example increase of the biomass of stem tuber, rhizome, bulb etc. of plant part.Particularly preferably be following each or multinomial increase: the length of the diameter of the number of the volume of the biomass of the root of increase, the root of increase, the root of increase, the root of increase and the root of increase.The output that term increases also comprises the increase of seed production.

Ding Yi term " seed production of increase " is used to represent to compare with corresponding wild type plant following each or multinomial increase herein: (i) the seed ultimate production of Zeng Jiaing, and it comprises that the increase of seed biomass (seed weight) and its can be the increases of every strain plant or the seed weight on single seed basis; The (ii) number of the every paniculiform flower of Zeng Jiaing (" Xiao Hua "); The (iii) number of the full seed of Zeng Jiaing; The (iv) seed size of Zeng Jiaing; (the v) seed volume of Zeng Jiaing; (the vi) single seed area of Zeng Jiaing (seed area); (the vii) single seeded length and/or the width of Zeng Jiaing; (the viii) harvest index of Zeng Jiaing, this exponential representation for can gather in the crops part for example the output of seed to the ratio of total biomass; (ix) the full rate of Zeng Jiaing (fill rate), (it is the overall number of the number of full seed divided by seed, multiply by 100 then); (x) thousand seed weight of Zeng Jiaing (TKW) is wherein calculated described thousand seed weight from the number of full seed and its gross weight of counting.The TKW that increases can be produced by seed size that increases and/or seed weight.The TKW that increases can be produced by the increase of embryo size and/or endosperm size.

With the corn is example, and the increase of output can show as in following one or multinomial: the increase of the length/diameter of the increase of the number of the fringe of every strain plant, tassel row number, a row grain number, grain weight, TKW, fringe, or the like.With the rice is example, and the output increase can show as one of following or multinomial increase: the increase of the number of the paniculiform number of every strain plant, every paniculiform small ear, the number of every paniculiform flower, the full rate of seed, the increase of TKW, or the like.The increase of output also can cause the structure that changes, or can take place because of the structure that changes.

By implementing the growth characteristics of the improvement that method of the present invention obtains, in the scope of the use that relates to CDC27, cause having the plant of the number seeds of increase.The number seeds that increases comprises with suitable control plant to be compared, the increase of the full rate of the increase of the increase of seed overall number and/or full seed number and/or seed (it multiply by 100 for the full seed number again divided by the overall number of seed), this increase can be the increase of every strain plant and/or the increase of per hectare or acre.With the corn is example, and the increase of number seeds is usually expressed as the increase, tassel row number of the spike number of every strain plant, the increase of row grain number, the increase of the full rate of seed, or the like.With the rice is example, and the increase of number seeds is usually expressed as the number of the paniculiform number of every strain plant, every paniculiform small ear, the increase of the number of every paniculiform flower (Xiao Hua) (it is expressed as the ratio of full seed number to the number of main panicle (primary panicles)), the increase of the full rate of seed.

Therefore the invention provides the method that is used for comparing with the number seeds of suitable control plant the number seeds that increases plant, this method comprises the expression of nucleic acids that preferentially increases coding CDC27 polypeptide in axis end meristematic tissue, and described polypeptide is at the NH of polypeptide ₂End regions has the TPR structural domain of at least one inactivation.

In method of the present invention relates to the scope of SYR, preferably implement the plant that described method causes producing the seed production with increase.More preferably, the seed production of increase comprise in (full) number seeds, seed gross weight, seed size, thousand seed weight, full rate and the harvest index one or more aspects separately with control plant increase Comparatively speaking.Therefore, according to the present invention, provide the method that is used to increase plant seed output, this method is included in the expression of nucleic acids of active and/or coding SYR polypeptide or its homologue that increase the SYR polypeptide in the plant.

In method of the present invention relates to the scope of FG-GAP, preferably carry out described method and cause producing output, more particularly the plant of the seed production of biomass of Zeng Jiaing and/or increase with increase.Preferably, the seed production of increase comprise in (full) number seeds, seed gross weight, seed size, thousand seed weight and the harvest index one or multinomial separately with control plant increase Comparatively speaking.Therefore, according to the present invention, provide to be used to increase plant biomass, increased the method for biomass and/or increase seed production especially, this method is included in the expression of nucleic acids of active and/or coding FG-GAP polypeptide or its homologue of regulating the FG-GAP polypeptide in the plant.

In method of the present invention related to the scope of CYP90B, the preferred output that increases comprised one or more aspects (each aspect is all for suitable control plant) of following aspect: the seed area of the HI of increase, the TKW of increase, increase and the seed length of increase.Therefore, according to the present invention, provide the control plant that is used for suitable to compare, increased the method for plant biomass, particularly seed production, this method is included in the non-constitutive expression that increases the nucleic acid of coding CYP90B polypeptide or its homologue in the plant.

In method of the present invention relates to the scope of AT hook transcription factor, increase the seed production in the monocotyledons.Therefore provide and be used for, compare with suitable control plant, increase the method for monocotyledonous seed production, this method comprises preferentially increases the expression of nucleic acids that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain in monocotyledonous endosperm tissue.

In method of the present invention related to the scope of DOF transcription factor, the preferred output that increases was the seed production that increases.According to a preferred feature of the present invention, provide to be used for, compare with the seed production of the control plant that suits, increase the method for plant seed output, this method is included in the expression of nucleic acids that increases encoding D OF transcription factor polypeptide in the plant.

In method of the present invention related to the scope of CKI, the growth characteristics of improvement was the seed production that increases.Therefore the invention provides and be used for, compare with suitable control plant, increase the method for the seed production of plant, this method comprises preferentially reduce endogenous CKI expression of gene in the endosperm tissue of plant.

Because the plant of improvement of the present invention has the output (seed production) of increase, therefore these plants may (its life cycle to small part period) show, compare the growth velocity of increase with the growth velocity of the corresponding wild-type plant of the respective stage that is in life cycle.The growth velocity that increases can be specific to one or more parts or the cell type (comprising seed) of plant, or can spread all over whole plants basically.Plant with growth velocity of increase can have shorter life cycle.The life cycle of plant is used in reference to: grow to the needed time in stage that plant produces the dried mature seed that is similar to parent material from the mature seed of doing.This life cycle can be subjected to the influence of for example early growth gesture of factor, growth velocity, flowering time and seed maturity speed.The increase of growth velocity can take place in life cycle in one or more stages of life cycle of plant or in whole plants basically.The growth velocity that increases in the plant commitment of life cycle can be reflected as enhanced growth potential.The increase of growth velocity can change the harvest cycle of plant, thereby permission plant ratio originally can late sowing kind and/or early harvest.If growth velocity obtains enough increases, it may allow to sow the seed (for example sow again fully and gather in the crops rice plant) of identical plant species again after sowing and results rice plant in a conventional vegetative period.Similarly, if growth velocity obtains enough increases, it may allow to sow different plant species (for example behind sowing and results rice, for example sowing and randomly gather in the crops soybean, potato or any other suitable plant again) again.Under the situation of some plants, the results of carrying out additional times from identical stock also may be fine.The harvest cycle that changes plant can cause every acre year biomass yield increase (increase of the number of times (in a year) that this can grow and gather in the crops owing to any specific plant).The increase of growth velocity also can allow transgenic plant to cultivate in than the wider geographic area of its wild type counterparts, because the region of crop culture restriction is normally by the adverse environment conditional decision of implantation time (season early) or harvest time (season in evening).If shorten harvest cycle then can avoid these disadvantageous conditions.Can be by determining growth velocity from growth curve (mapping obtains to growth experiment) the various parameters of derivation, this type of parameter can be: T-Mid (plant reaches its maximum sized 50% time spent) and T-90 (plant reaches its maximum sized 90% time spent), or the like.The term of Shi Yonging " flowering time " should refer to that seed germination began to the time period of blooming between the beginning herein.

The enforcement of the inventive method has produced the plant of the growth velocity with increase.

Therefore, according to the present invention, provide the method for the growth velocity that is used to increase plant, this method is included in to be increased the active of SYR polypeptide or its homologue and/or increases this proteic expression of nucleic acids of coding in the plant.

According to the present invention, the method for the growth velocity that is used to increase plant is provided, this method is included in the plant regulates the active of (preferred increasing) FG-GAP polypeptide or its homologue and/or regulates (preferred increasing) this proteic expression of nucleic acids of coding.

According to the present invention, the method for the growth velocity that is used to increase plant is provided, this method is included in the non-constitutive expression that increases the nucleic acid of coding CYP90B polypeptide or its homologue in the plant.

According to the present invention, the method for the growth velocity that is used to increase plant is provided, this method is included in the expression of nucleic acids that increases encoding D OF transcription factor polypeptide in the plant.

According to the present invention, provide to be used for, with respect to suitable control plant, increase the method for the growth velocity of plant, this method comprises preferentially minimizing endogenous cell cyclin-dependent kinase inhibitor (CKI) expression of gene in the endosperm tissue of plant.

No matter plant is in still is exposed to different coercing under the non-stress conditions, all will take place to compare the increase of output and/or seed production and/or growth velocity with control plant.Plant is reacted to being exposed to coerce by growth is next more lentamente usually.Under serious stress conditions, plant even may stop growing fully.On the other hand, slight coercing is defined as plant herein and is exposed to this and coerces the back, do not cause plant to stop growing and lose any of ability who regrows and coerce.On meaning of the present invention, slightly coercing the plant that causes being coerced compares with the control plant under non-stress conditions, growth reduces less than 40%, 35% or 30%, preferred less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or lower.Because the progress of agricultural practice (irrigation, fertilising, pesticide-treated) can not run into serious coercing usually in the crop plants of cultivation.Therefore, the feature of normally not expecting on the agricultural by the growth that weakens of slight stress-inducing.Slightly coercing can be that the typical case that plant may touch coerces.These are coerced can be plant contact to biological and/or abiotic (environment) of every day coerce.Typical abiotic or environment-stress comprise by the temperature that atypical heat or cold/freezing temperature causes coerce, salt stress, water coerce that (arid or too much water), anoxic are coerced, chemical toxicity and oxidative stress.Abiotic stress can be to coerce (special because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress that causes.Chemical substance also can cause abiotic stress (for example mineral substance of too high or too low concentration or nutrition).Biology is coerced normally by pathogenic agent coercing of causing of bacterium, virus, fungi and insect for example.The term of Shi Yonging " non-stress conditions " is meant that these envrionment conditionss exceed plant weather condition every day that can run into and permission plant optimum growh and other abiotic conditions indistinctively herein.Those skilled in the art will understand the normal edaphic condition and the weather condition in given geographical position.

In method of the present invention related to the scope of SYR, the enforcement of described method caused producing the plant of the abiotic stress resistance with increase.As reporting among the people such as Wang (Planta (2003) 218:1-14), abiotic stress causes influencing the growth of plant unfriendly and a series of morphology, physiology, biological chemistry and the molecule of productivity changes.Known arid, salinity, extreme temperature and oxidative stress connect each other, and can induce the growth and the infringement of cell by similar mechanism.For example, arid and/or salinification mainly show as osmotic stress, thereby cause the destruction of homeostasis and ion distribution in the cell.Oxidative stress often is accompanied by high temperature or low temperature, salinity or drought stress, and it can cause functional and structural proteic sex change.As a result of, these different environment-stress usually activate similar cellular signal transduction pathways and cell response, for example rise of the generation of stress protein, antioxidant, accumulation and cessation of growth cessation that can miscible solute.

Because different environment-stress can activate similar approach, only be not confined to drought stress so the present invention should not regard as with illustrating of carrying out of drought stress (using in the scope of SYR polypeptide and its coding nucleic acid the present invention relates to), reflect that prevailingly SYR polypeptide or its homologue participate in a kind of performance of abiotic stress and more should regard as.In addition, also can implement the inventive method under the non-stress conditions or under slight drought condition, thereby produce the plant of comparing growth characteristics (output of increase especially) with corresponding wild type or other control plants with improvement.

" dialogue (crosstalk) " people (2003) Plant Physiol 133:1755-1767 such as () Rabbani that between drought stress and high-salt stress, has special high level according to reports.Therefore, clearly SYR polypeptide or its homologue with and purposes in giving plant arid resistance, can be used for the infringement that protective plant is avoided multiple other abiotic stress too.Similarly, clearly SYR albumen (as defined here) and its are given the purposes of salt tolerance in plant, also can be used for the infringement that protective plant is avoided multiple other abiotic stress.In addition, people such as Rabbani (2003, Plant Physiol 133:1755-1767) are reported in and have similar stress resistance and the molecular mechanism of replying between dicotyledons and the monocotyledons.Therefore, method of the present invention advantageously can be used for any plant.

Ding Yi term " abiotic stress " is used in reference to that water coerces that (because arid or too much water cause), anoxic are coerced herein, salt stress, temperature are coerced (because hot, cold or ice-cold temperature cause), chemical toxicity is coerced with oxidative stress in any one or more coerce.According to an aspect of the present invention, abiotic stress is an osmotic stress, is selected from that water is coerced, salt stress, oxidative stress and ion coerce.Preferably, to coerce be drought stress to water.The term salt stress is not limited to common salt (NaCl), can also be NaCl, KCl, LiCl, MgCl ₂, CaCl ₂Or the like in any one or more salt.

The resistance to abiotic stress that increases finds expression in the plant biomass that increases under the abiotic stress condition.In the scope of the purposes that the present invention relates to SYR polypeptide and its coding nucleic acid, the output of such increase can comprise one or more following aspects (each aspect all with corresponding wild type plant Comparatively speaking): the root of the harvest index of the number of every paniculiform flower of the full seed number of increase, the seed ultimate production of increase, increase, the full rate of seed of increase, increase, the thousand seed weight of increase, increase is grown or the root diameter of increase.

The enforcement of the inventive method produces the plant of the abiotic stress resistance with increase.The enforcement of the inventive method has caused when growing under the non-stress conditions or under slight drought condition, compare with the corresponding wild-type plant of under comparable condition, growing or other control plants, have the plant of the growth characteristics (the particularly gesture of emerging of output of Zeng Jiaing and/or increase (or early growth gesture)) of improvement.

According to the present invention, the method for the abiotic stress resistance that is used to increase plant is provided, this method is included in the expression of nucleic acids of regulating coding SYR polypeptide or its homologue in the plant.According to an aspect of the present invention, abiotic stress be selected from that water is coerced, salt stress, oxidative stress and ion one or more the osmotic stress in coercing.Preferably, to coerce be drought stress to water.

The present invention also provides the method for the abiotic stress resistance that is used for improving plant, and this method is included in the activity that increases SYR albumen or its homologue in the plant.

In method of the present invention relates to the scope of DOF transcription factor, can under slight drought condition, implement present method, to produce the plant of comparing output with suitable control plant with increase.As reporting among the people such as Wang (Planta (2003) 218:1-14), abiotic stress causes influencing unfriendly a series of morphology, physiology, biological chemistry and the molecule of plant-growth and productivity to change.Known arid, salinity, extreme temperature and oxidative stress be connect each other and can induce the growth and the infringement of cell by similar mechanism.People such as Rabbani (Plant Physiol (2003) 133:1755-1767) have described " dialogue " that there be special high level between drought stress and the high-salt stress.For example, arid and/or salinification mainly show as osmotic stress, thereby cause the homeostasis in the cell and the destruction of ion distribution.Oxidative stress (being accompanied by high temperature or low temperature, salinity or drought stress usually) can cause functional and structural proteic sex change.Therefore, these different environment-stress usually activate similar cellular signal transduction pathways and cell response, for example rise of the generation of stress protein, antioxidant, accumulation and cessation of growth cessation that can miscible solute.

When the enforcement of the inventive method causes growing under slight drought condition, compare, have the plant of the output of increase with the suitable control plant of under comparable condition, growing.Therefore, according to the present invention, provide the method that is used to be increased in the output of growing plants under the slight drought condition, this method is included in the expression of nucleic acids that increases encoding D OF transcription factor polypeptide in the plant.

Advantageously can in any plant, improve the growth characteristics of above-mentioned improvement.In method of the present invention related to the scope of purposes of AT hook transcription factor, described method can be used for monocotyledons.

The term of Shi Yonging " plant " comprises ancestors of complete plant, plant and the part of offspring and plant herein, comprise seed, branch, stem, leaf, root (comprising stem tuber), flower and tissue and organ, wherein aforementioned each all comprise goal gene/nucleic acid or the genetic modification in goal gene/nucleic acid.Term " plant " also comprises vegetable cell, suspension culture, callus, embryo, meristem zone, gametophyte, sporophyte, pollen and sporule, and is same, wherein above-mentioned each all comprise goal gene/nucleic acid.

The plant that is particularly useful for method of the present invention comprises all plants that belong to vegitabilia (Viridiplantae) superfamily, particularly monocotyledons and dicotyledons, comprise the feed or the leguminous forage that are selected from following plants, ornamental plant, alimentary crop, tree or shrub: Acer (Acer spp.), Actinidia (Actinidia spp.), Abelmoschus (Abelmoschus spp.), Agropyron (Agropyronspp.), allium (Allium spp.), Amaranthus (Amaranthus spp.), pineapple (Ananascomosus), Anona (Annona spp.), celery (Apium graveolens), Arachis (Arachis spp), Jack-fruit belongs to (Artocarpus spp.), officinalis (Asparagusofficinalis), Avena (Avena spp.) (oat (Avena sativa) for example, wild swallow grass (Avenafatua), than praising oat (Avena byzantina), Avena fatua var.sativa, hybrid oat (Avena hybrida)), carambola (Averrhoa carambola), wax gourd (Benincasahispida), Brazil's chestnut (Bertholletia excelsea), beet (Beta vulgaris), Btassica (Brassica spp) (colea (Brassica napus) for example, overgrown with weeds blue or green (Brassica rapassp.) [canola, oilseed rape (oilseed rape), rape (turnip rape)]), Cadabafarinosa, tea (Camellia sinensis), Canna generalis Bailey (Canna indica), Capsicum (Capsicum spp.), Carex elata, papaya (Carica papaya), Carissa macrocarpa (Carissa macrocarpa), hickory (Carya spp.), safflower (Carthamustinctorius), Castanea (Castanea spp.), cultivation witloof (Cichorium endivia), Cinnamomum (Cinnamomum spp.), watermelon (Citrullus lanatus), Citrus (Citrus spp.), cocoanut (Cocos spp.), Coffea (Coffea spp.), taro (Colocasia esculenta), Cola spp, coriander (Coriandrum sativum), Corylus (Corylus spp.), hawthorn (Crataegus spp.), Stigma Croci (Crocus sativus), Cucurbita (Cucurbita spp.), Cucumis (Cucumis spp.), Lay Cirsium (Cynara spp.), Daucus carota L. (Daucus carota), beggar-ticks (Desmodium spp.), longan (Dimocarpus longan), Wild yam (Dioscorea spp.), Diospyros (Diospyros spp.), Echinochloa (Echinochloa spp.), oil palm belongs to (Elaeis) (oil palm (Elaeis guineensis) for example, America oil palm (Elaeis oleifera)) Finger-millet (Eleusine coracana), loquat (Eriobotrya japonica), haw son (Eugeniauniflora), Fagopyrum (Fagopyrum spp.), Fagus (Fagus spp.), Fructus Fici (Ficus carica), Fortunella (Fortunella spp.), Fragaria (Fragaria spp.), ginkgo (Ginkgo biloba), Glycine (Glycine spp.) (soybean (Glycine max for example, Soja hispida or Soja max)), upland cotton (Gossypium hirsutum), Helianthus (Helianthus spp) (for example Sunflower Receptacle (Helianthus annuus)), tawny daylily (Hemerocallisfulva), hibiscus (Hibiscus spp.), Hordeum (Hordeum spp.) (for example barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), white walnut (Juglans spp.), lettuce ((Lactuca sativa), Lathyrus (Lathyrus spp.), Lens culinaris (Lens culinaris), flax (Linum usitatissimum), lichee (Litchi chinensis), Lotus (Lotusspp.), Guangdong sponge gourd (Luffa acutangula), lupinus (Lupinus spp.), Luzulasylvatica, tomato belongs to (Lycopersicon spp.) (tomato (Lycopersiconesculentum) for example, tomato (Lycopersicon lycopersicum), Lycopersicon pyriforme), sclerderm Macroptilium (Macrotyloma spp.), Malus (Malus spp.), recessed edge Malpighia coccigera (Malpighia emarginata), horse rice apple (Mammea americana) Mango fruit (Mangifera indica), cassava (Manihot spp.), sapota (Manilkara zapota), alfalfa (Medicago sativa), Melilotus sweetclover (Melilotus spp.), Mentha (Menthaspp.), Momordica (Momordica spp.), black mulberry (Morus nigra), Musa (Musaspp.), Nicotiana (Nicotiana spp.), Olea (Olea spp.), Opuntia (Opuntiaspp.), bird foot Macroptilium (Ornithopus spp.), Oryza (Oryza spp.) (rice (Oryzasativa) for example, broad-leaved rice (Oryza latifolia)), millet (Panicum miliaceum), Purple Granadilla (Passiflora edulis), Selinum pastinaca (Pastinaca sativa), Persea (Persea spp.), parsley (Petroselinum crispum), Phaseolus (Phaseolus spp.), the thorn certain herbaceous plants with big flowers belongs to ((Phoenixspp.), Physalis (Physalis spp.), Pinus (Pinus spp.), Pistacia vera (Pistaciavera), Pisum (Pisum spp.), annual bluegrass belongs to (Poa spp.), Populus (Populus spp.), Prosopis (Prosopis spp.), Prunus (Prunus spp.), Psidium (Psidium spp.), pomegranate (Punica granatum), Ussurian pear (Pyrus communis), oak belongs to (Quercusspp.), radish (Raphanus sativus), rheum rhabarbarum (Rheum rhabarbarum), currant belongs to (Ribes spp.), castor-oil plant (Ricinus communis), rubus (Rubusspp.), saccharum (Saccharum spp.), Sambucus (Sambucus spp.), rye (Secale cereale), flax belongs to (Sesamum spp.), sinapsis alba belongs to (Sinapis sp.), Solanum (Solanum spp.) (potato (Solanum tuberosum) for example, red eggplant (Solanumintegrifolium) or tomato (Solanum lycopersicum)), Chinese sorghum (Sorghum bicolor), spinach belongs to (Spinacia spp.), Syzygium (Syzygium spp.), Tagetes (Tagetesspp.), tamarind (Tamarindus indica), cocoa (Theobroma cacao), Clover (Trifolium spp.), Triticosecale rimpaui, Triticum (Triticum spp.) (common wheat (Triticum aestivum) for example, durum wheat (Triticum durum), duckbill wheat (Triticum turgidum), Triticum hybernum, Macha wheat (Triticum macha) (Triticummacha), common wheat (Triticum sativum or Triticum vulgare)), little Flower of Chinese Globeflower (Tropaeolum minus), nasturtium (Tropaeolum majus), genus vaccinium (Vacciniumspp.), Vetch (Vicia spp.), Vigna (Vigna spp.), Viola odorata (Violaodorata), Vitis (Vitis spp.), Zea mays (Zea mays), the living wild rice in natural pond (Zizaniapalustris), zizyphus (Ziziphus spp.), or the like.

Preferably, plant is for example soybean, Sunflower Receptacle, Canola, alfalfa, Semen Brassicae campestris, cotton, tomato, potato or a tobacco of crop plants.Preferred in addition, plant is a monocotyledons, for example sugarcane.More preferably plant is a cereal, for example rice, corn, wheat, barley, grain (millet), rye, Chinese sorghum or oat.

When method of the present invention related to the purposes of AT hook transcription factor, monocotyledons was a cereal, for example rice, corn, sugarcane, wheat, barley, grain, rye, Chinese sorghum, grass or oat.

Definition

Polypeptide

Term " polypeptide " and " protein " are used interchangeably herein, are meant the amino acid that the polymer form with any length exists.Term " polynucleotide ", " nucleotide sequence ", " nucleotide sequence " are used interchangeably herein, are meant the Nucleotide that the polymer form with any length exists, ribonucleotide or deoxyribonucleotide or both combinations.

Control plant

The selection of suitable control plant is the conventional part of experimental program, the plant that it can comprise corresponding wild type plant or not have goal gene accordingly.Control plant plant common and to be assessed is identical plant species or even identical kind.Control plant can also be the invalid zygote (nullizygote) of plant to be assessed." control plant " of Shi Yonging not only is meant complete plant herein, but also refers to plant part, comprises the part of seed and seed.

Increase, improve

Term " increase ", " improvement " or " raising " are used interchangeably herein, be used to represent compare with corresponding wild type or other control plants as definition herein, height at least 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% output and/or growth.

Hybridization

Ding Yi term " hybridization " is a homology complementary nucleotide sequence annealed process each other basically wherein herein.Can be fully in solution (promptly two complementary nucleic acids are all in solution) carry out crossover process.Can also be fixed to matrix at one of complementary nucleic acid for example hybridizes under the situation of magnetic bead, sepharose 4B or any other resin.Crossover process can also a nucleic acid in complementary nucleic acid be fixed to solid support for example Nitrocellulose or nylon membrane or be fixed under for example siliceous glass support situation of (latter is called nucleic acid array or microarray or nucleic acid chip) by for example light version typography take place.In order to allow hybridization to take place, usually nucleic acid molecule is carried out thermally denature or chemical modification, be 2 strands and/or eliminate hairpin structure or thereby two strands unwind from other secondary structures of single-chain nucleic acid.The stringency of hybridization is subjected to for example influence of temperature, salt concn, ionic strength and hybridization buffer composition of condition.

In the situation of nucleic acid hybridization experimental example such as Southern and Northern hybridization, " tight hybridization conditions " is sequence-dependent and different under the varying environment parameter with " tight hybridization wash conditions ".Those skilled in the art will know that can hybridization and washing process in various parameters that change and that will keep or change the stringency condition.

T _mBe under ionic strength of determining and pH, the temperature the during probe hybridization of 50% target sequence and coupling fully.T _mThe based composition and the length that depend on solution condition and probe.For example, longer sequence specific hybrid under higher temperature.Be lower than T _mAbout 16 ℃ to 32 ℃ obtain maximum hybridization speed.The existence of monovalent cation has reduced the Coulomb repulsion between 2 nucleic acid chains in the hybridization solution, thereby promotes crossbred to form; This effect is a visible for the na concn that reaches 0.4M.Methane amide reduces the melting temperature(Tm) of DNA-DNA and DNA-RNA duplex, and per 1% methane amide reduces by 0.6 to 0.7 ℃, and the adding of 50% methane amide allows to hybridize under 30 to 45 ℃, although hybridization speed will reduce.Base-pair mismatch reduces the thermostability of hybridization speed and duplex.Fifty-fifty with for big probe, per 1% base mispairing, T _mReduce about 1 ℃.The type that depends on crossbred can use following formula to calculate T _m:

DNA-DNA crossbred (Meinkoth and Wahl, Anal.Biochem., 138:267-284,1984):

T _m=81.5 ℃+16.6xlog[Na ⁺] ^a+ 0.41x%[G/C ^b]-500x[L ^c] ^-1-0.61x% methane amide

DNA-RNA or RNA-RNA crossbred:

T _m=79.8+18.5 (log ₁₀[Na ⁺] ^a)+0.58 (%G/C ^b)+11.8 (%G/C ^b) ²-820/L ^cFew DNA or few RNA ^dCrossbred:

For＜20 Nucleotide: T _m=2 (l _n)

For 20-35 Nucleotide: T _m=22+1.46 (l _n)

^aOr for other monovalent cations, but in the 0.01-0.4M scope, be accurately only.

^bBe accurately only to the %GC in 30% to 75% scope.

^cThe length of the duplex in the L=base pair

^dOligo, oligonucleotide; l _n, the useful length of primer=(number of G/C)+(number of A/T).

Attention: for per 1% methane amide, T _mReduce about 0.6 to 0.7 ℃, yet the existence of 6M urea reduces T _mAbout 30 ℃.

The specificity of hybridization is the function of post-hybridization washing normally.For eliminating the background that causes by non-specific hybridization, with the salts solution washing sample of dilution.The key factor of such washing comprises the ionic strength and the temperature of whole washing soln: salt concn is low more and wash temperature is high more, and then Xi Di stringency is high more.Be generally equal to or be lower than the hybridization stringency and implement wash conditions.Usually, it is as implied above to be used for the suitable stringent condition of nucleic acid hybridization test or gene amplification detection method.Also can select more tight or not too tight condition.Usually, low stringency condition is chosen as the pyrolysis chain point (T of bit sequencing row under ionic strength of determining and pH _m) low about 50 ℃.Medium stringency condition is that temperature compares T _mLow 20 ℃, high stringency condition is that temperature compares T _mLow 10 ℃.For example, the stringency condition is the same with for example condition A-L at least tight stringent condition; The stringency condition that reduces is the same with for example condition M-R at least tight stringency condition.Can use the arbitrary technology in many known technology (for example with comprising proteinic solution closing membrane, in hybridization buffer, adding allos RNA, DNA and SDS and handle) to control non-specific binding with the RNA enzyme.

The example of hybridization and wash conditions is listed in table 1:

Table 1:

" crossbred length " is the expection length that the nucleic acid of hybridization takes place.When making the nucleic acid hybridization of known array, can determine crossbred length by aligned sequences and evaluation conserved regions described herein.

(1 * SSPE is 0.15M NaCl, 10mM NaH to SSPE ₂PO ₄, and 1.25mM EDTA, pH7.4) SSC (1 * SSC is 0.15M NaCl and 15mM Trisodium Citrate) in alternative hybridization and the lavation buffer solution; Finished after scouring 15 minutes in hybridization.Hybridization and washing can comprise 5 * Denhardt ' s reagent, 0.5-1.0%SDS, 100 μ g/ml salmon sperm DNA sex change, fragmentation, 0.5% trisodium phosphate extraly and be no more than 50% methane amide.

^*Tb-Tr: be used to expect that length should be than the melting temperature(Tm) T of crossbred less than the hybridization temperature of the crossbred of 50 base pairs _mLow 5-10 ℃; Determine T according to above-mentioned formula _m

^±The present invention also comprises with the replacement nucleic acid of PNA or modification any one or a plurality of DNA or RNA hybridization mating partner.

In order to define the level of stringency, can be easily with reference to people such as Sambrook (2001) Molecular Cloning:a laboratory manual, the 3rd edition, Cold Spring HarborLaboratory Press, CSH, New York or with reference to Current Protocols in MolecularBiology, John Wiley ﹠amp; Sons, N.Y. (1989).

T-DNA activates label (Activation Tagging)

T-DNA activates label people Science (1992) 1350-1353 such as () Hayashi and relates to T-DNA (comprising promotor (also can be translational enhancer or intron) usually) is inserted with such configuration in the upstream or downstream 10kb of coding region of the genome area of goal gene or gene, and described configuration makes promotor can instruct this by the expression of gene of target.Usually, interrupt, gene is belonged under the control of promotor of new importing by of the regulation and control of the natural promoter of the gene of target to its expression.Usually promotor is embedded T-DNA.This T-DN is for example infected the insertion Plant Genome by Agrobacterium (Agrobacterium) at random, thus near the gene overexpression the T-DNA that causes inserting.The transgenic plant of gained are owing to the expression of crossing of the gene of the close promotor that imports shows the dominant phenotype.The promotor that is imported into can be any promotor that can instruct gene to express in the biology (being plant under this situation) of expectation.For example, composing type, organize preference type promotor, cell type preference type and inducible promoter all to be suitable for T-DNA to activate.

The local mutating technology (TILLING) of directional induction genome

TILLING (the local mutating technology of directional induction genome) is the induced-mutation technique that is used to produce and/or identify and/or finally separate the variant nucleic acid of mutagenesis.TILING also allows to select to have the plant of this mutation variants.The variant of these sudden changes even can show the activity higher than the gene of its natural form.TILLING combines high-density mutagenesis and high-throughput screening method.The step of carrying out usually among the TILLING is: (a) EMS mutagenesis (Redei GP and Koncz C (1992) InMethods in Arabidopsis Research, Koncz C, Chua NH, Schell J, eds.Singapore, World Scientific Publishing Co, pp.16-82; People such as Feldmann, (1994) In Meyerowitz EM, Somerville CR, eds, Arabidopsis.Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, pp 137-172; LightnerJ and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods onMolecular Biology, the 82nd volume .Humana Press, Totowa, NJ, pp 91-104); (b) DNA of individual preparation and merging (pooling); (c) pcr amplification in purpose zone; (d) sex change and annealing so that heteroduplex can form; (e) DHPLC wherein merges existing in of heteroduplex in the thing and detects in the color atlas and be extra peak; (f) evaluation of mutated individual; (g) order-checking of sudden change PCR product.The method that is used for TILLING is well-known (people such as McCallum, (2000) Nat Biotechnol 18:455-457 in this area; By Stemple (2004) Nat Rev Genet 5 (2): 145-50 summarizes).

Directed mutagenesis

Can use directed mutagenesis to produce the variant of SYR nucleic acid.Can obtain several method and carry out directed mutagenesis; The most frequently used method that is based on PCR (Current Protocols in Molecular Biology.Wiley Eds. Http:// www.4ulr.com/products/currentprotocols/index.html).

Transposon mutagenesis

Transposon mutagenesis is based on the induced-mutation technique of the insertion of transposon in gene, and this mutagenesis often causes gene knockout.This technology has been used for the several plant species, comprise rice (people such as Greco, PlantPhysiol, 125,1175-1177,2001), corn (people such as McCarty, Plant J.44,52-61,2005) and Arabidopsis (Parinov and Sundaresan, Curr.Opin.Biotechnol.11,157-161,2000).

Orthogenesis

Orthogenesis or gene reorganization (gene shuffling) (thereby produce variant nucleic acid or its part by DNA reorganization repeatedly with suitable then screening and/or selection, or polypeptide or its homologue with biologic activity of change) form (people such as Castle, (2004) Science304 (5674): 1151-4; United States Patent (USP) 5,811,238 and 6,395,547).

Homologous recombination

Homologous recombination allows at the select location of determining selected nucleic acid quiding gene group.Homologous recombination is that routine is used for for example standard technique of yeast or liver moss sword-like leave Rhodobryum (Physcomitrella) of unicellular lower eukaryote in the bio-science.Not only at model plant (people (1990) EMBO J 9 (10) such as Offringa: 3077-84) but also at crop plants rice (people (2002) Nat Biotech 20 (10): 1030-4 such as Terada for example; Iida and Terada (2004) Curr Opin Biotech15 (2): 132-8) described the method that is used for carrying out homologous recombination plant.Target nucleic acid (it can be any nucleic acid or the variant that herein defines) needs the directed special genes seat that arrives.Target nucleic acid can be that the allelotrope that is used to replace the improvement of native gene maybe can be the extra importing outside native gene.

Homologue

Proteinic " homologue " comprise compare with the protein of described unmodified have amino-acid substitution, disappearance and/or insertion and have the similar biologic activity of the protein of the unmodified of originating and peptide, oligopeptides, polypeptide, protein and the enzyme of functionally active to them.In order to produce such homologue, the available proteinic amino acid of other amino-acid substitutions with similar quality (tendency of for example similar hydrophobicity, wetting ability, antigenicity, formation or destruction αLuo Xuanjiegou or βZhe Die structure).The preservative replacement table in this area be well-known (referring to for example Creighton (1984) Proteins.W.H.Freeman and Company and below table 2).

Lineal homologue and collateral line homologue

Term " homologue " comprises direct line (orthologous) homologous sequence and collateral line (paralogous) homologous sequence,, comprises the homologue of two kinds of special shapes of the evolution notion of the ancestral relationship that is used to describe gene that is.

Term " collateral line homologue " relates to the gene that causes in the homogenic species gene group of collateral line to be doubled.Can easily identify the collateral line homologue by analyzing at carry out BLAST from one group of sequence of the species identical with search sequence.

Term " lineal homologue " relates to the homologous gene in the different biologies that cause owing to species formation.Can easily find lineal homologue in the dicotyledons species for example by carrying out so-called mutual blast research.This can be undertaken by a blast (comprising the BLAST analysis that for example can implement search sequence (for example, SEQ ID NO:1 or SEQ ID NO:2) at any sequence library at the obtainable ncbi database of the public that http://www.ncbi.nlm.nih.gov. finds).When starting from nucleotide sequence, can use BLASTN or TBLASTX (use standard default), when starting from protein sequence, can use BLASTP or TBLASTN (use standard default).Can randomly filter BLAST result.Then filtering result or unfiltered result's full length sequence is analyzed (the 2nd BLAST) (when search sequence is SEQ ID NO:1 or SEQ ID NO:2, the 2nd blast will carry out at the rice sequence) at return BLAST from the sequence of the biology that search sequence derived from.The result who compares first and second BLAST then.If from identical species, be accredited as the collateral line homologue so from the high-level hit event of the 2nd blast and search sequence; If high-level hit event and search sequence from different species, are accredited as lineal homologue so.High-level hit event is the hit event with low E value.The E value is low more, mark remarkable more (or in other words, the probability that chances on this hit event is low more).The calculating of E value is known in this area.Under the situation of extended familys, can use ClustalW, make up then in abutting connection with tree (neighbourjoining tree), show the cluster of genes involved and identify lineal homologue and collateral line homologue with help.

Homologue can be the form of proteinic " displacement variant ", and promptly wherein at least one residue of aminoacid sequence has been removed and has inserted different residues in its position.The normally single residue form of amino-acid substitution, but depend on that the function restriction that places on the polypeptide also can take place by cluster; Insert the rank of normally about 1 to 10 amino-acid residue.Preferably, amino-acid substitution comprises conservative amino acid replacement.Can not the displacement of cannot not carrying out conservatively under the very vital situation in above-mentioned amino acid character.The conservative substitution table can obtain in this area easily.Following table has provided the example of conservative amino acid replacement.

Table 2: the example of conservative amino acid replacement

Residue	Conservative substitution	Residue	Conservative substitution
Residue	Conservative substitution	Residue	Conservative substitution	Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln	Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln	Asn	Gln；His	Met	Leu；Ile
Asp	Glu	Phe	Met；Leu；Tyr	Asn	Gln；His	Met	Leu；Ile
Asp	Glu	Phe	Met；Leu；Tyr	Gln	Asn	Ser	Thr；Gly
Cys	Ser	Thr	Ser；Val	Gln	Asn	Ser	Thr；Gly
Cys	Ser	Thr	Ser；Val	Glu	Asp	TPR	Tyr
Gly	Pro	Tyr	TPR；Phe	Glu	Asp	TPR	Tyr
Gly	Pro	Tyr	TPR；Phe	His	Asn；Gln	Val	Ile；Leu
Ile	Leu，Val			His	Asn；Gln	Val	Ile；Leu

Homologue can also be the form of proteinic " insertion variant ", promptly wherein one or more amino-acid residues is imported predetermined position in the protein.Insertion can comprise in N end and/or C end fusions and the single or multiple amino acid whose sequence inserts.Usually, the insertion in the aminoacid sequence will be littler than N or the fusion of C end, be the rank of about 1 to 10 residue.The example of N or C end fusion rotein or peptide comprises the binding domains of the activating transcription factor that is used for yeast two-hybrid system or activation structure territory, bacteriophage coat protein, (Histidine)-6-label, glutathione S-transferring enzyme-label, A albumen, maltose binding protein, Tetrahydrofolate dehydrogenase, Tag100 epi-position, c-myc epi-position, FLAG

-peptide, lacZ, CMP (calmodulin binding peptide), HA epi-position, C albumen epi-position and VSV epi-position.

For the homologue of proteinic " disappearance variant " form is characterised in that one or more amino acid is removed from protein.

Can use the peptide synthetic technology known in this area for example solid-phase peptide is synthetic and wait or prepare proteinic amino acid variant by recombinant DNA processing ease ground.Being used to operate dna sequence dna knows in this area with the method that produces proteinic displacement, insertion or disappearance variant.For example, the technology that is used for generation replacement mutation on the predetermined position of DNA is known to those skilled in the art, it comprises M13 mutagenesis, T7-Gen vitro mutagenesis (USB, Cleveland, OH), QuickChange directed mutagenesis (Stratagene, San Diego, CA), directed mutagenesis or other directed mutagenesis schemes of PCR mediation.

Derivative

" derivative " is to compare with the aminoacid sequence of the form (promptly not experiencing posttranslational modification) of the natural generation of protein (for example, the protein shown in SEQ ID NO:2) to comprise that modify and/or the non-natural modified amino acid residue natively polypeptide or protein.Proteinic " derivative " comprises and compares polypeptide or the protein change that can comprise natural generation, glycosylated, acidylate, prenylation or the amino-acid residue that non-natural takes place with the aminoacid sequence of the form of the natural generation of polypeptide.Derivative is compared with its aminoacid sequence of originating and also can be comprised one or more non-aminoacid replacement bases, for example be bonded to reporter molecules or other parts of aminoacid sequence covalency or non-valency, for example in conjunction with the reporter molecules of the detection that helps aminoacid sequence and the amino-acid residue that takes place with respect to the non-natural of the proteinic aminoacid sequence of natural generation.

The alternative splicing variant

The term of Shi Yonging " alternative splicing variant " comprises wherein and excises, replaces or add selected intron and/or exon or wherein shortened or increased the variant of the nucleotide sequence of intron herein.This type of variant is the variant that wherein proteinic biologic activity obtains keeping, and can obtain such variant by the proteinic function fragment of selective retention.But can find or this type of splice variant of artificial preparation at occurring in nature.The method that is used to produce this type of splice variant is known in this area.

Allele variant

The natural existence of allele variant, method of the present invention comprise this type of natural allelic application.Allele variant comprises single nucleotide polymorphism (SNP) and little insertion/deletion polymorphism (SmallInsertion/Deletion Polymorphism) (INDEL).The size of INDEL is usually less than 100bp.SNP and INDEL have formed one group of maximum sequence variants in the polymorphism strain system of the natural generation of most of biologies.

Promotor

Term " controlling element ", " control sequence " and " promotor " all are used interchangeably at this, and it broadly refers to realize the regulation and control nucleotide sequence of the expression of the sequence that is connected with them.Above-mentioned term comprises that deriving from classical eukaryotic gene group gene transcription regulating and controlling sequence (comprises the necessary TATA box of transcription initiation accurately, have or do not have CCAAT box sequence) and response growth and/or outside stimulus or change the extra controlling element (that is, upstream activating sequence, enhanser and silencer) of genetic expression in tissue-specific mode.This term also comprises the transcription regulating nucleotide sequence of classical prokaryotic gene, and it can comprise-35 box sequences and/or-10 box transcription regulating nucleotide sequences in this case.Term " controlling element " also comprises synthetic fusion molecule or the derivative of giving, activating or strengthen the expression of nucleic acid molecule in cell, tissue or organ.The term of Shi Yonging " effectively connect " is meant the functional connection between promoter sequence and the goal gene herein, and what promoter sequence just can initial goal gene like this transcribes.

Promotor can be an inducible promoter, i.e. response is grown, chemistry, environment or physical stimulation and induce or increase transcription initiation.

Organize preference type or tissue-specific promoter be can be preferentially some for example organize leaf, root, seed tissue etc. or even in specific cell initial promotor of transcribing.

Ding Yi term " composing type " is meant main at least one tissue or organ and mainly in any life stage expression promoter of plant herein.Preferred described promotor is mainly expressed in whole plants.

The example of other constitutive promoters is shown in following table 3.

Table 3: the example of constitutive promoter

Gene source	Reference
Gene source	Reference	Actin muscle	McElroy etc., Plant Cell, 2:163-171,1990
CAMV?35S	Odell etc., Nature, 313:810-812,1985	Actin muscle	McElroy etc., Plant Cell, 2:163-171,1990
CAMV?35S	Odell etc., Nature, 313:810-812,1985	CaMV?19S	Nilsson etc., Physiol.Plant.100:456-462,1997
GOS2	De Pater etc., Plant J Nov; 2 (6): 837-44,1992, WO2004/065596	CaMV?19S	Nilsson etc., Physiol.Plant.100:456-462,1997
GOS2		Ubiquitin	Christensen etc., Plant Mol.Biol.18:675-689,1992
The rice cyclophilin	Buchholz etc., Plant Mol Biol.25 (5): 837-43,1994	Ubiquitin	Christensen etc., Plant Mol.Biol.18:675-689,1992
The rice cyclophilin	Buchholz etc., Plant Mol Biol.25 (5): 837-43,1994	Corn H3 histone	Lepetit etc., Mol.Gen.Genet.231:276-285,1992
Clover H3 histone	Plant Mol.Biol.11:641-649 such as Wu, 1988	Corn H3 histone	Lepetit etc., Mol.Gen.Genet.231:276-285,1992
Clover H3 histone	Plant Mol.Biol.11:641-649 such as Wu, 1988	Actin muscle 2	An etc., Plant are (1) J.10; 107-121,1996
34S?FMV	Sanger?et?al.，Plant.Mol.Biol.，14，1990：433-443	Actin muscle 2	An etc., Plant are (1) J.10; 107-121,1996
34S?FMV	Sanger?et?al.，Plant.Mol.Biol.，14，1990：433-443	The Rubisco small subunit	US?4,962,028
OCS	Leisner(1988)Proc?Natl?Acad?Sci?USA?85(5)：2553	The Rubisco small subunit	US?4,962,028
OCS	Leisner(1988)Proc?Natl?Acad?Sci?USA?85(5)：2553	SAD1	Jain etc., Crop Science, 39 (6), 1999:1696
SAD2	Jain etc., Crop Science, 39 (6), 1999:1696	SAD1	Jain etc., Crop Science, 39 (6), 1999:1696
SAD2	Jain etc., Crop Science, 39 (6), 1999:1696	Nos	Shaw etc. (1984) Nucleic Acids Res.12 (20): 7831-7846
V-ATPase	WO?01/14572	Nos	Shaw etc. (1984) Nucleic Acids Res.12 (20): 7831-7846
V-ATPase	WO?01/14572	Super promotor	WO?95/14098
G box protein matter	WO?94/12015	Super promotor	WO?95/14098

Table 4: the example of non-constitutive promoter

Gene source and title	Expression pattern	Reference
Gene source and title	Expression pattern	Reference	Rice RP6	Endosperm-specific	People such as Wen (1993) Plant Physiol 101 (3): 1115-6
The kafirin of Chinese sorghum	Endosperm-specific	People such as DeRose (1996) PlantMolec Biol 32:1029-35	Rice RP6	Endosperm-specific	People such as Wen (1993) Plant Physiol 101 (3): 1115-6
The kafirin of Chinese sorghum	Endosperm-specific	People such as DeRose (1996) PlantMolec Biol 32:1029-35	The zein of corn	Endosperm-specific	People such as Matzke (1990) PlantMol Biol 14 (3): 323-32
Rice oleosin 18kDa	Embryo (and aleuron) is specific	People such as Chuang (1996) J Biochem 120 (1): 74-81	The zein of corn	Endosperm-specific	People such as Matzke (1990) PlantMol Biol 14 (3): 323-32
Rice oleosin 18kDa	Embryo (and aleuron) is specific	People such as Chuang (1996) J Biochem 120 (1): 74-81	Rice oleosin 16kDa	Embryo (and aleuron) is specific	People such as Chuang (1996) J Biochem 120 (1): 74-81
Soybean β-conglycinin	Embryo	People such as Chiera (2005) Plant Molec Biol 56 (6): 895-904	Rice oleosin 16kDa	Embryo (and aleuron) is specific	People such as Chuang (1996) J Biochem 120 (1): 74-81
Soybean β-conglycinin	Embryo		Rice Wsi18	Whole seed	People such as Joshee (1998) Plant CellPhysiol 39 (1): 64-72.
Rice	Whole seed	People such as Sasaki (2002) NCBI accession number BAA85411	Rice Wsi18	Whole seed
Rice	Whole seed	People such as Sasaki (2002) NCBI accession number BAA85411	Rice OSH1	Early stage stem end meristematic tissue	People such as Sato (1996) Proc Natl Acad Sci 93 (15): 8117-8122
Rice Rcc2	Root-specific	People such as Xu (1995) Plant MolBiol 27 (2): 237-48	Rice OSH1	Early stage stem end meristematic tissue
Rice Rcc2	Root-specific	People such as Xu (1995) Plant MolBiol 27 (2): 237-48	Rice Rcc3	Root-specific	People such as Xu (1995) Plant MolBiol 27 (2): 237-48
Arabidopsis Pyk10	Root-specific	People such as Nitz (2001) Plant Sci161 (2): 337-346	Rice Rcc3	Root-specific	People such as Xu (1995) Plant MolBiol 27 (2): 237-48

Table 5: the example of early stage stem end meristematic tissue promotor

Gene source	Gene family	Plant origin	Reference
Gene source	Gene family	Plant origin	Reference	OSH1	KNOX family	1 class homeobox	Rice	People such as-Matsuoka, people such as (1993) Plant Cell 5:1039-1048-Sato, (1996) PNAS 93:8117-8122
Knotted1	KNOX family	1 class homeobox	Corn	OSH1	KNOX family	1 class homeobox	Rice		People such as Hake, (1989) EMBOJournal 8:15-22
Knotted1	KNOX family	1 class homeobox	Corn	KNAT1	KNOX family	1 class homeobox	Arabidopis thaliana	People such as Lincoln, (1994) PlantCell 6:1859-1876	People such as Hake, (1989) EMBOJournal 8:15-22

Oskn2	KNOX family	1 class homeobox	Rice	People such as Postma-Haarsma, (1999) Plant Mol Biol 39 (2): 257-71
Oskn2	KNOX family	1 class homeobox	Rice		Oskn3	KNOX family	1 class homeobox	Rice	People such as Postma-Haarsma, (1999) Plant Mol Biol 39 (2): 257-71

Table 6: the example that is used for endosperm specificity promoter of the present invention

Gene source	Expression pattern	Reference
Gene source	Expression pattern	Reference	Wheat LMW and HMW glutenin 1	Endosperm	Mol?Gen?Genet?216：81-90，1989；NAR?17：461-2，1989.
Wheat α, beta, gamma-gliadine	Endosperm	EMBO?3:1409-15，1984.	Wheat LMW and HMW glutenin 1	Endosperm	Mol?Gen?Genet?216：81-90，1989；NAR?17：461-2，1989.
Wheat α, beta, gamma-gliadine	Endosperm	EMBO?3:1409-15，1984.	Barley Itr1 promotor	Endosperm
Barley B1, C, D, hordein	Endosperm	Theor?Appl?Gen?98：1253-62，1999；Plant?J?4：343-55，1993；Mol?Gen Genet?250：750-60，1996.	Barley Itr1 promotor	Endosperm
Barley B1, C, D, hordein	Endosperm		Barley DOF	Endosperm	People such as Mena, The Plant Journal, 116 (1): 53-62,1998.
blz2	Endosperm	EP99106056.7	Barley DOF	Endosperm
blz2	Endosperm	EP99106056.7	The synthetic promotor	Endosperm	People such as Vicente-Carbajosa, Plant J.13:629-640,1998.
Paddy prolamine NRP33	Endosperm	People such as Wu, Plant Cell Physiology39 (8) 885-889,1998.	The synthetic promotor	Endosperm	People such as Vicente-Carbajosa, Plant J.13:629-640,1998.
Paddy prolamine NRP33	Endosperm		Rice alpha-globulin Glb-1	Endosperm	People such as Wu, Plant Cell Physiology39 (8) 885-889,1998.
Rice alpha-globulin REB/OHP-1	Endosperm	People such as Nakase, Plant Mol.Biol.33:513-522,1997.	Rice alpha-globulin Glb-1	Endosperm
Rice alpha-globulin REB/OHP-1	Endosperm	People such as Nakase, Plant Mol.Biol.33:513-522,1997.	Rice ADP-glucose PP	Endosperm	Trans?Res?6：157-68，1997.
Corn ESR gene family	Endosperm	Plant?J?12：235-46，1997.	Rice ADP-glucose PP	Endosperm	Trans?Res?6：157-68，1997.
Corn ESR gene family	Endosperm	Plant?J?12：235-46，1997.	Chinese sorghum γ-kafirin	Endosperm	PMB?32：1029-35，1996.

Table 7: the example that is used for seed specific promoters of the present invention

Gene source	Expression pattern	Reference
Gene source	Expression pattern	Reference	The seed-specific gene	Seed	Simon waits the people, Plant Mol.Biol.5:191,1985; Scofield waits the people, J.Biol.Chem. 262:12202,1987; Baszczynski waits the people, Plant Mol.Biol.14:633,1990.

Brazil's nut albumin	Seed	Pearson waits the people, Plant Mol.Biol.18:235-245,1992.
Brazil's nut albumin	Seed	Pearson waits the people, Plant Mol.Biol.18:235-245,1992.	Legumin	Seed	Ellis waits the people, Plant Mol.Biol.10:203-214,1988.
Gluten (rice)	Seed	Takaiwa waits the people, Mol.Gen.Genet.208:15-22,1986; Takaiwa waits the people, FEBSLetts.221:43-47,1987.	Legumin	Seed	Ellis waits the people, Plant Mol.Biol.10:203-214,1988.
Gluten (rice)	Seed		Zein	Seed	People such as Matzke, Plant Mol Biol, 14 (3): 323-32,1990.
napA	Seed	Stalberg waits the people, Planta 199:515-519,1996.	Zein	Seed	People such as Matzke, Plant Mol Biol, 14 (3): 323-32,1990.
napA	Seed	Stalberg waits the people, Planta 199:515-519,1996.	Wheat LMW and HMW glutenin-1	Endosperm	Mol?Gen?Genet?216:81-90，1989；NAR17：461-2，1989.
Wheat SPA	Seed	People such as Albani, Plant Cell, 9:171-184,1997.	Wheat LMW and HMW glutenin-1	Endosperm	Mol?Gen?Genet?216:81-90，1989；NAR17：461-2，1989.
Wheat SPA	Seed	People such as Albani, Plant Cell, 9:171-184,1997.	Wheat α, beta, gamma-gliadine	Endosperm	EMBO?3：1409-15，1984.
Barley Itr1 promotor	Endosperm		Wheat α, beta, gamma-gliadine	Endosperm	EMBO?3：1409-15，1984.
Barley Itr1 promotor	Endosperm		Barley B1, C, D, hordein	Endosperm	Theor?Appl?Gen?98：1253-62，1999；Plant?J?4：343-55，1993；Mol?GenGenet?250：750-60，1996.
Barley DOF	Endosperm	People such as Mena, The Plant Journal, 116 (1): 53-62,1998.	Barley B1, C, D, hordein	Endosperm
Barley DOF	Endosperm		blz2	Endosperm	EP99106056.7
The synthetic promotor	Endosperm	People such as Vicente-Carbajosa, Plant J.13:629-640,1998.	blz2	Endosperm	EP99106056.7
The synthetic promotor	Endosperm	People such as Vicente-Carbajosa, Plant J.13:629-640,1998.	Paddy prolamine NRP33	Endosperm	People such as Wu, Plant Cell Physiology 39 (8) 885-889,1998.
Rice alpha-globulin Glb-1	Endosperm	People such as Wu, Plant Cell Physiology 39 (8) 885-889,1998.	Paddy prolamine NRP33	Endosperm
Rice alpha-globulin Glb-1	Endosperm		Rice OSH1	Embryo	People such as Sato, Proc.Natl.Acad.Sci.USA, 93:8117-8122,1996.
Rice alpha-globulin REB/OHP-1	Endosperm	People such as Nakase, Plant Mol.Biol.33:513-522,1997.	Rice OSH1	Embryo
Rice alpha-globulin REB/OHP-1	Endosperm	People such as Nakase, Plant Mol.Biol.33:513-522,1997.	Rice ADP-glucose PP	Endosperm	Trans?Res?6：157-68，1997.
Corn ESR gene family	Endosperm	Plant?J?12：235-46，1997.	Rice ADP-glucose PP	Endosperm	Trans?Res?6：157-68，1997.
Corn ESR gene family	Endosperm	Plant?J?12：235-46，1997.	Chinese sorghum γ-kafirin	Endosperm	PMB?32：1029-35，1996.

KNOX	Embryo	People such as Postma-Haarsma, Plant Mol.Biol.39:257-71,1999.
KNOX	Embryo		The rice oleosin	Embryo and aleuron	People such as Wu, J.Biochem., 123:386,1998.
Sunflower Receptacle oil albumen	Seed (embryo and dry seeds)	People such as Cummins, Plant Mol.Biol.19:873-876,1992.	The rice oleosin	Embryo and aleuron	People such as Wu, J.Biochem., 123:386,1998.

The terminator sequence

Term " terminator " comprises control sequence, and described control sequence is that the end in transcription unit is the dna sequence dna that 3 ' processing of primary transcript and polyadenylation and the termination of transcribing provide signal.Other controlling element can comprise transcriptional enhancer and translational enhancer.Those skilled in the art will know that and to be suitable for carrying out terminator of the present invention and enhancer sequence.This type of sequence is known or can easily obtains by those skilled in the art.

Selective marker

The term of herein mentioning " selectable marker gene " comprises any following gene, and described gene pairs is expressed its cell and given phenotype, thereby helps to identify and/or select with nucleic acid construct transfection of the present invention or cell transformed.Suitable mark can be selected from gives microbiotic or Herbicid resistant, the mark that imports new metabolism proterties or allow range estimation to select.The example of selectable marker gene comprises giving (for example to be provided Basta microbiotic (hpt of for example nptII of phosphorylation Xin Meisu and kantlex, or phosphorylation Totomycin), weedicide ^TMThe bar of resistance; The aroA or the gox of the resistance of resistance glyphosate are provided) resistance gene or the gene (for example allowing plant to use the manA of seminose as sole carbon source) of metabolism proterties is provided.The visual marking gene cause color (β-glucuronidase for example, GUS), the formation of luminous (for example luciferase) or fluorescence (green fluorescent protein, GFP and its derivative).

Transform

The term of herein mentioning " conversion " comprises exogenous polynucleotide is transferred to host cell, and regardless of the method that is used to shift.Available genetic constructs of the present invention transforms the plant tissue that can carry out clonal propagation (taking place or the embryo generation by organ) subsequently, then from the complete plant of its regeneration.The specific tissue of selecting can change, and this depends on the clonal propagation system of the specific species that can obtain to be used for and be suitable for most being transformed.The example organization target (for example comprises leaf dish, pollen, embryo, cotyledon, hypocotyl, megagametophyte (megagametophyte), callus, existing meristematic tissue, apical meristem, axillalry bud and root meristematic tissue) and inductive meristematic tissue (for example, cotyledon meristematic tissue and hypocotyl meristematic tissue).Can be with polynucleotide instantaneous or stably import host cell, it can keep the nonconformity state for example as plasmid.Selectively, can be integrated into host genome.With method known to those skilled in the art the plant transformed cell of gained is used to the plant transformed of regenerating then.

The conversion of plant species is quite conventional technology now.Advantageously, can use the arbitrary method in several method for transformation that goal gene is imported suitable ancester cell.Method for transformation comprise the chemical substance, DNA of use, electroporation, the picked-up of increase dissociative DNA of liposome to direct injection, the particle gun bombardment (particle gun bombardment) of plant, use the conversion and the microinjection of virus or pollen.Method is optional from the calcium/polyoxyethylene glycol method (Krens, people such as F.A., (1982) Nature296, the 72-74 that are used for protoplastis; People such as Negrutiu I (1987) Plant Mol Biol 8:363-373), the electroporation of protoplastis (people (1985) Bio/Technol 3 such as Shillito R.D., 1099-1102), the microinjection (people such as Crossway A, (1986) Mol.GenGenet 202:179-185) to the vegetable material; The microparticle bombardment of DNA or RNA bag quilt people such as (, (1987) Nature 327:70) Klein TM; Use the infection of (nonconformable) virus etc.Preferably by agriculture bacillus mediated conversion, use any method of knowing that is used for the rice conversion, the method of in one of following documents and materials, describing for example: disclosed European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199:612-617,1996); People such as Chan (Plant Mol Biol 22 (3): 491-506,1993), people such as Hiei (Plant J 6 (2): 271-282,1994) (its disclosure intactly is incorporated herein by reference) produce transgenosis rice plant.Under the situation that corn transforms, preferred method is that (Nat.Biotechnol 14 (6): 745-50 as people such as Ishida, 1996) or the method for describing among people's (PlantPhysiol 129 (1): 13-22,2002) (its disclosure is intactly introduced herein as a reference) such as Frame.

Usually after conversion, just, select vegetable cell or cell cohort by the existence of one or more marks of the expressive gene of plant coding that moves with the goal gene corotation, afterwards the material regeneration that transforms is become complete plant.

After DNA shifts and regenerates, can for example use Southern to analyze the conversion plant of existence, copy number and/or genomic tissue assessment supposition with regard to goal gene.Selectively or in addition, can use Northern and/or Western to analyze the expression level of the new DNA that imports of (two kinds of technology are known to those skilled in the art) monitoring.

Can be by several different methods for example by clonal propagation or classical breeding technique, the plant transformed that breeding is produced.For example, can make the selfing of the first-generation (or T1) plant transformed, select the s-generation (or T2) transformant isozygoty then, can further breed the T2 plant by the breeding technique of classics then.

The inverting biological that is produced can be taked various ways.For example, they can be the mosaics of transformant and no transformed cells; Clone's property transformant (for example, all cells is comprised expression cassette through transforming); That transform and grafting unconverted tissue (for example, in plant, the stock of the conversion of grafting to the unconverted scion).

The detailed description of seed production conditioning agent (SYR)

Can increase the proteic activity of SYR by the level that increases the SYR polypeptide.Selectively, when the level of SYR does not change, or even when the proteic level of SYR reduces, activity also can increase.This can occur in when for example by producing mutant or when selecting to have more active variant and change the inwardness of polypeptide than wild-type.

Ding Yi term " SYR albumen or its homologue " is meant about 65 to about 200 amino acid whose polypeptide herein, this polypeptide (i) comprises the be rich in leucic structural domain similar to leucine zipper in proteinic C end half part, (ii) this (guards primitive 1a for having sequence YFS before being rich in leucic structural domain, SEQ ID NO:6) or YFT (conservative primitive 1b, SEQ ID NO:7) or YFG (conservative primitive 1c, SEQ ID NO:8) or YLG (conservative primitive 1d, SEQ ID NO:9) tripeptides, (iii) this is conservative primitive 2 ((V/A/I) LAFMP (T/S), SEQID NO:10) after being rich in leucic structural domain.Preferably, conservative primitive 2 is (A/V) LAFMP (T/S), should conservative primitive be VLAFMPT most preferably." SYR albumen or its homologue " preferably also has the conservative C-terminal peptide with conservative primitive 3 (SYL or PYL, SEQ ID NO:11) ending.The length that is rich in leucic structural domain of SYR albumen or its homologue is about 38 to 48 amino acid (just in time start from after the conservative primitive 1 and just end at and guard before the primitive 2), and it comprises at least 30% leucine.The structural domain that is rich in Leu preferably has and leucine zipper primitive (L-X ₆-L-X ₆-L-X ₆-L, wherein X ₆Be the sequence of 6 continuous amino acids) similar primitive.The proteic preferred embodiment of SYR is represented by SEQ ID NO:2, provided the general introduction of its structural domain in Fig. 1.Should be pointed out that term " SYR albumen or its homologue " does not comprise the ARGOS albumen (SEQ ID NO:26) from Arabidopis thaliana.

More preferably, SYR albumen has 2 membrane spaning domains, and wherein proteic N end parts and C end parts are positioned at inside, and the part between two membrane spaning domains is positioned at the outside.

Selectively, the proteic homologue of SYR has and the amino acid 27% shown in the SEQ ID NO:2 by the preferred sequence that increases progressively at least, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complete sequence identity, condition is that homologous protein comprises above-mentioned conservative primitive 1 (a, b, c or d), 2 and 3 and be rich in leucic structural domain.Use the overall comparison algorithm for example program GAP (GCG WisconsinPackage, Accelrys) the Needleman Wunsch algorithm (preferably using default parameter) in can be determined complete sequence identity.

Can use special database to identify that various structural domains in the SYR albumen, described database are SMART (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Schultz, 5857-5864 for example; People such as Letunic (2002) Nucleic Acids Res 30,242-244; Http:// smart.embl-heidelberg.de/), InterPro (people such as Mulder, (2003) Nucl.Acids.Res.31,315-318; Http:// www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and itsfunction in automatic sequence interpretation. (In) ISMB-94; The 2nd international conference minutes Altman R. of molecular biology intelligence system, Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAIPress, Menlo Park; People such as Hulo, Nucl.Acids.Res.32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (people such as Bateman, Nucleic Acids Research 30 (1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/).

The method that is used for the search of SYR homologue and evaluation is fully within those skilled in the art's limit of power.These class methods comprise uses the algorithm of knowing in this area that is used for sequence alignment or comparison, with the sequences of the SEQ ID NO:1 of computer-reader form or 2 expressions with can public database for example in MIPS (http://mips.gsf.de/), GenBank really good (http://www.ncbi.nlm.nih.gov/Genbank/index.html) or the EMBL nucleotide sequence database (http://www.ebi.ac.uk/embl/index.html) sequence of acquisition compare, described algorithm is GAP (Needleman and Wunsch, J.Mol.Biol.48 for example; 443-453 (1970)), BESTFIT (uses local homology's algorithm (Advances inApplied Mathematics 2 of Smith and Waterman; 482-489 (1981))), BLAST (Altschul, S.F., Gish, W., Miller, W., Myers, E.W.﹠amp; Lipman, D.J., J.Mol.Biol.215:403-410 (1990)), FASTA and TFASTA (W.R.Pearson and D.J.Lipman Proc.Natl.Acad.Sci.USA 85:2444-2448 (1988)).Be used to carry out the software that BLAST analyzes and pass through the public acquisition of American National biotechnology information center (NCBI).

Membrane spaning domain length is about 15 to 30 amino acid, and is made up of the hydrophobic residue that forms the α spiral usually.Usually based on hydrophobicity predict they (people such as Klein for example, Biochim.Biophys.Acta 815,468,1985; Or people such as Sonnhammer, J.Glasgow, T.Littlejohn, F.Major, R.Lathrop, D.Sankoff and C.Sensen compile, Proceedingsof the Sixth International Conference on Intelligent Systems for MolecularBiology, the 175-182 page or leaf, Menlo Park, CA, 1998.AAAI Press.).

The proteinic example that drops under the definition of " SYR polypeptide or its homologue " is listed in the Table A of embodiment 1, and it comprises from different monocotyledonss for example rice (SEQ ID NO:2, SEQ ID NO:12 and SEQ ID NO:13), corn (SEQ ID NO:14 and SEQ ID NO:44), wheat (SEQID NO:15), barley (SEQ ID NO:16), sugarcane (SEQ ID NO:17 and SEQ ID NO:18), Chinese sorghum SEQ ID NO:19) sequence; With from the dicotyledons sequence of Arabidopsis (SEQID NO:20 and SEQ ID NO:21), grape (SEQ ID NO:22), oranges and tangerines (SEQ ID NO:23) or tomato (SEQ ID NO:24 and SEQ ID NO:25) for example.Can predict, the structural domain that is rich in Leu is very important for proteinic function, therefore has the structural domain that is rich in Leu but do not have the albumen of guarding primitive 1 or 2 to can be used for method of the present invention equally; Proteinic example like this is shown in SEQ ID NO:34 and 35.

Be appreciated that term " SYR polypeptide or its homologue " is not limited to sequence or the listed homologue of SEQ ID NO:12 to SEQ ID NO:25 that SEQ ID NO:2 represents, comprise the standard that is rich in leucic structural domain as defined above but satisfy; Or have and all can be suitable for method of the present invention to about 200 amino acid whose polypeptide any about 65 of the sequence identity of the sequence at least 38% of SEQ ID NO:2, being conservative tripeptides primitive 1 (a, b, c or d) before the wherein said structural domain, is conservative primitive 2 and preferably in addition conservative primitive 3 afterwards.

In another embodiment, the invention provides and be selected from following isolating SYR albumen:

(a) polypeptide of expression among the SEQ ID NO 44,

(b) has the polypeptide of following aminoacid sequence, described aminoacid sequence by the preferred sequence that increases progressively have with SEQ ID NO 44 in the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of expression

(c) (a) or (b) in the proteic derivative of definition.

The sequence of being represented by SEQ ID NO:43 is still unknown so far as the gene of coding SYR.Therefore the invention provides isolated nucleic acid sequences, this sequence comprises

(i) nucleotide sequence of representing by SEQ ID NO:43, or its complementary strand;

(ii) the encode nucleotide sequence of the aminoacid sequence represented by SEQ ID NO:44;

(iii) can (preferably under stringent condition) with above (i) or the nucleotide sequence of nucleic acid array hybridizing (ii), this hybridization sequences optimized encoding SYR albumen;

(iv) as the nucleic acid of the allele variant of (i) or nucleotide sequence (ii);

(v) as the nucleic acid of the splice variant of (i) or nucleotide sequence (ii);

(vi) have (i) or the nucleotide sequence of the sequence identity of the sequence 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% of definition (ii) or 99%.

Can be by in rice, being expressed in the activity that SYR albumen under the GOS2 promotor control or its homologue are measured SYR albumen or its homologue, this causes when comparing with corresponding wild type plant, has the seed production of increase and does not have the plant that flowering time postpones.Can measure the increase of this seed production in several modes, for example with the form of the increase of seed gross weight, full seed number or harvest index.

SYR albumen or its homologue are by SYR nucleic acid/genes encoding.Therefore the term " SYR nucleic acid/gene " of definition is any nucleic acid/gene of SYR albumen as defined above or its homologue of encoding herein.

The example of SYR nucleic acid includes but not limited to the nucleic acid by any expression among SEQ ID NO:1, SEQ ID NO:27 to SEQ ID NO:32, SEQ ID NO:36 to 42 and the SEQ ID NO:44.The also tabulation of mentioned nucleic acid in the Table A referring to embodiment 1.

SYR nucleic acid/gene and its variant can be suitable for putting into practice method of the present invention.Variant SYR nucleic acid/gene comprise SYR nucleic acid/gene part and/or can with the nucleic acid of SYR nucleic acid/gene recombination.

Ding Yi term " part " is meant the fragment of coding about 65 to the DNA of about 200 amino acid whose polypeptide herein, wherein said polypeptide comprises and is rich in leucic structural domain as defined above, being conservative tripeptides primitive 1 (a, b, c or d) before this structural domain, is conservative primitive 2 and preferably in addition conservative primitive 3 afterwards.Preferably, part comprises one or more conservative as defined above primitives.Can for example prepare this part by in SYR nucleic acid, producing one or more disappearances.Can use this part maybe their fusions to other codings (or non-coding) sequence can be made up several active albumen for example to produce with isolating form.When merging to other encoding sequences, the polypeptide that produces after the translation of gained can be bigger than the size of being predicted at this SYR fragment.Preferably, part is the part by the nucleic acid of any expression among SEQ IDNO:1, SEQ ID NO:27 to SEQ ID NO:32, SEQ ID NO:36 to SEQ ID NO:42 and the SEQ ID NO:44.Most preferably the part of nucleic acid is shown in SEQ ID NO:1.

The another kind of variant of SYR nucleic acid/gene be can be under the stringency condition that reduces (preferably under stringent condition) and the nucleic acid of the SYR nucleic acid/gene recombination of definition above, this hybridization sequences encodes about 65 to about 200 amino acid whose polypeptide, this polypeptide comprises and is rich in leucic structural domain as defined above or has sequence identity to the sequence at least 38% of SEQ ID NO:2, be conservative primitive 1 (a, b, c or d) before the wherein said structural domain, and be conservative primitive 2 and preferably in addition conservative primitive 3 afterwards.

Preferably, hybridization sequences be can with the nucleic acid of representing by SEQ ID NO:1, SEQ ID NO:27 to SEQ IDNO:32, SEQ ID NO:36 to SEQ ID NO:42 and SEQ ID NO:44 or with the sequence of the part hybridization of arbitrary sequence of above-mentioned sequence.Most preferably hybridization sequences can be hybridized SEQID NO:1.Term " hybridization " is the hybridization that this paper defines.

SYR nucleic acid or its variant can derive from any natural or artificial source.Can be from microbe-derived for example yeast or fungi or from plant, algae or animal (comprising the people) source isolating nucleic acid/gene or its variant.Can in composition and/or genome environment, modify this nucleic acid by the manual operation of having a mind to from its natural form.Nucleic acid is plant origin preferably, and it can derive from identical plant species (for example identical with its plant to be imported) or from different plant species.Can be from the unifacial leaf species, preferably from Gramineae (Poaceae), more preferably from the rice isolating nucleic acid.More preferably, the SYR separate nucleic acid is represented from rice and by SEQ ID NO:1, and the SYR aminoacid sequence is represented by SEQ ID NO:2.

Can regulate the expression of nucleic acids of coding SYR polypeptide or its homologue by importing genetic modification (preferably in the locus of SYR gene).The locus of Ding Yi gene is used to represent genome area herein, and this zone comprises the 10kb upstream or the downstream of goal gene and coding region.

Can for example import genetic modification by any (or a plurality of) method in the following method or by the nucleic acid that in plant, imports and express coding SYR polypeptide or its homologue, described method is: T-DNA activation, TILLING, directed mutagenesis,, transposon mutagenesis, orthogenesis and homologous recombination.Defined aforesaid method at title in the part of " definition " herein.After importing genetic modification, the expression of nucleic acids of just encode SYR polypeptide or its homologue changes, and selects step, and wherein the change in this expression causes having the plant of the seed production of increase.

T-DNA activation, TILLING, directed mutagenesis, transposon mutagenesis and orthogenesis are the examples that can produce the technology of new allelotrope and SYR variant.

A preferred method that is used for importing genetic modification (described modification does not need to be arranged in the locus of SYR gene in this case) is to import and express the coding SYR polypeptide of definition herein or the nucleic acid of its homologue plant.The nucleic acid of plant to be imported can be total length nucleic acid maybe can be as defined above the part or hybridization sequences.

Defined proteinic " homologue " at title for the part of " definition " in this manual.SYR polypeptide or its homologue can be derivatives.About the definition of term " derivative ", be entitled as the part of " definition " referring to the acceptance of the bid of this specification sheets.

SYR polypeptide or its homologue can be by the alternative splicing variant codings of SYR nucleic acid/gene.Defined term " alternative splicing variant " in " definition " part.Preferred splice variant is the splice variant of coding about 65 to the nucleic acid of about 200 amino acid whose polypeptide, described polypeptide comprises and is rich in leucic structural domain as defined above or has sequence identity to the sequence at least 38% of SEQ ID NO:2, be conservative tripeptides primitive 1 (a, b, c or d) before the wherein said structural domain, and be conservative primitive 2 and preferably in addition conservative primitive 3 afterwards.The splice variant of being represented by SEQ ID NO:1, SEQ ID NO:27 to SEQ ID NO:32, SEQ ID NO:36 to SEQ ID NO:42 and SEQ ID NO:44 is further preferred.The splice variant that SEQ ID NO:1 represents is most preferred.

Homologue also can be by the allele variant of the nucleic acid of coding SYR polypeptide or its homologue, optimized encoding about 65 is to the allele variant coding of the nucleic acid of about 200 amino acid whose polypeptide, described polypeptide comprises and is rich in leucic structural domain as defined above or has sequence identity to the sequence at least 38% of SEQ ID NO:2, be conservative tripeptides primitive 1 (a, b, c or d) before the wherein said structural domain, and be conservative primitive 2 and preferably in addition conservative primitive 3 afterwards.More preferably, by the allele variant of the coding SYR polypeptide of any expression among SEQ IDNO:1 or SEQ ID NO:12 to the SEQ ID NO:25.Most preferably, the allele variant of coding SYR polypeptide is the variant of being represented by SEQ IDNO:1.Defined term " allele variant " in " definition " part.

According to a preferred aspect of the present invention, can expect the expression of the increase of SYR nucleic acid or its variant.Be used to increase method existing lot of documents instruction in this area of the expression of gene or gene product.It comprises, for example by the use of crossing expression, transcriptional enhancer or translational enhancer of suitable promoters driven.Can import isolating nucleic acid as promotor or enhancer element in the suitable position (usually in the upstream) of the polynucleotide of non-allos form to raise the expression of SYR nucleic acid or its variant.For example, can by the sudden change, the disappearance and/or the displacement change in vivo endogenesis promoter (referring to, Kmiec, United States Patent (USP) 5,565,350; People such as Zarling PCT/US93/03868), or can import vegetable cell with isolating promotor with appropriate direction and distance (with respect to gene of the present invention), with the expression of controlling gene.The method that is used to reduce the expression of gene or gene product is known in this area.

If the expectation expression of polypeptides, the 3 ' end that is desirably in the polynucleotide encoding district usually comprises polyadenylation region.Polyadenylation region can derive from natural gene, from multiple other plant gene or from T-DNA.3 ' the end sequence that adds for example can derive from nopaline synthase or octopine synthase gene or selectively from another plant gene, or more preferably from any other eukaryotic gene.

Also can in the encoding sequence of 5 ' non-translational region or part encoding sequence, add intron sequences to be increased in the ripe courier's who accumulates in the cytosol amount.But be presented at and comprised the montage intron in the transcription unit of plant and animal expression construct and can make expression of gene on mRNA and protein level, increase nearly 1000 times, Buchman and Berg, Mol.Cell biol.8:4395-4405 (1988); People such as Callis, Genes Dev.1:1183-1200 (1987).In the time of near placing 5 of transcription unit ' end, this intron enhancement of genetic expression is maximum usually.The purposes of corn intron A dh1-

S introne

1,2 and 6, Bronze-1 intron is known in this area.Usually referring to, The MaizeHandbook, 116 chapters, Freeling and Walbot, Eds., Springer, N.Y. (1994).

The present invention also provides the importing of the nucleotide sequence that helps to be used for the inventive method and/or the genetic constructs and the carrier of expression.

Therefore, provide gene construct, it comprises:

(i) SYR nucleic acid or its variant that defines hereinbefore;

(ii) can drive one or more control sequences of the nucleotide sequence expression of (i); Randomly

(iii) transcription termination sequence;

Condition is the nucleic acid sequences to proteins that gene construct does not comprise coding SEQ ID NO:26.

Can use recombinant DNA technology well known to those skilled in the art to make up the construct that is used for method of the present invention.Gene construct can be inserted the carrier that is suitable for being transformed into plant and is suitable for expression goal gene in transformant, the commercially available acquisition of this carrier.

The carrier that use comprises aim sequence (that is the nucleic acid of coding SYR polypeptide or its homologue) transforms plant.Aim sequence effectively is connected with one or more control sequences (at least with promotor).Term " controlling element ", " control sequence " and " promotor " all are used interchangeably in this article, and title defines for the part of " definition " in this manual.

Advantageously, can use the expression of the promoters driven nucleotide sequence of any kind.Preferably, SYR nucleic acid or its functional variant effectively are connected with constitutive promoter.Preferably, can be preferentially in whole plants the constitutive promoter of express nucleic acid have the express spectra suitable with the GOS2 promotor.More preferably, constitutive promoter has and the identical express spectra of rice GOS2 promotor, most preferably, can be preferentially in whole plants the promotor of express nucleic acid be GOS2 promotor (SEQ IDNO:5) from rice.

Should be understood that the scope of application of the present invention is not limited to the SYR nucleic acid of being represented by SEQ ID NO:1, the scope of application of the present invention also is not limited to the SYR expression of nucleic acids by the GOS2 promoters driven.The selectable constitutive promoter that is used for method of the present invention is high mobility group protein (high mobility group protein, HMGP) promotor (SEQ ID NO:33).The example that also can be used for driving other constitutive promoters of SYR expression of nucleic acid is shown in the table 3 of title for the part of " definition ".

The construct that randomly, also one or more terminator sequences can be used for plant to be imported.Defined term " terminator " in " definition " part.

Genetic constructs of the present invention also can comprise in order to keep and/or duplicate necessary replication orgin sequence in specific cell type.An example is need be with in additive type genetic elements (for example plasmid or clay molecule) the maintenance genetic constructs in bacterial cell.Preferred replication orgin includes but not limited to f1-ori and colE1.

Genetic constructs can randomly comprise selectable marker gene, as the selectable marker gene in the definition of " definition " part.

The present invention also comprises can be by the plant of method acquisition of the present invention.Therefore the invention provides can be by the plant of method acquisition of the present invention, and described plant has imported SYR nucleic acid or its variant as defined above therein.

The present invention also provides the method for the transgenic plant that are used to produce the seed production with increase, and this method is included in and imports and express SYR nucleic acid or its variant as defined above in the plant.

More particularly, the invention provides the method for the transgenic plant that are used to produce the seed production with increase, this method comprises:

(i) in plant or vegetable cell, import and express SYR nucleic acid or its variant and

(ii) culturing plants cell under the condition that promotes plant-growth and growth;

Condition is that SYR nucleic acid or its variant are not the proteic nucleotide sequences of coding SEQ ID NO:26.

Nucleic acid directly can be imported vegetable cell or import plant itself (comprising the tissue, organ or any other part that import plant).According to a preferred feature of the present invention, preferably nucleic acid is imported plant by transforming.Defined term " conversion " in " definition " part.

The present invention clearly extends to any vegetable cell or plant and its all plant parts and the propagulum that produces by any method described herein.The present invention further extends to and comprises that the former generation conversion that produces by any method in the aforesaid method or the offspring of cells transfected, tissue, organ or whole plants, unique requirement are that this offspring shows genotype and/or identical genotype and/or the phenotypic characteristic of phenotypic characteristic with parent's generation in the method for the invention.The present invention also comprises the host cell that comprises isolating SYR nucleic acid or its variant.Preferred host cell of the present invention is a vegetable cell.The part gathered in the crops that the present invention also extends to plant for example but is not limited to seed, leaf, fruit, flower, stem culture, rhizome, stem tuber and bulb.The invention still further relates to the product of the part gathered in the crops that is directed to such plant, for example dried granulated feed (pellet) or dry powder, oil, fat and lipid acid, starch or protein.

The present invention also comprises the purposes of SYR nucleic acid or its variant and the purposes of SYR polypeptide or its homologue.

Such purposes relates to the growth characteristics of improveing plant, particularly aspect the raising seed production.Seed production can comprise one or more following aspects: the harvest index of the seed gross weight of increase, the full seed number of increase, full rate and increase.

SYR nucleic acid or its variant, or SYR polypeptide or its homologue can be used for the procedure of breeding, in the described procedure of breeding, identify can with the dna marker of SYR gene or its variant genetic linkage (genetically link).SYR nucleic acid/gene or its variant, or SYR polypeptide or its homologue can be used for defining molecule marker.This DNA or protein labeling can be used for the procedure of breeding has the seed production of increase with selection plant then.SYR gene or its variant can for example be the nucleic acid by any expression among SEQ ID NO:1, SEQ ID NO:27 to SEQ ID NO:32, SEQ ID NO:36 to SEQ ID NO:42 and the SEQ ID NO:44.

The allele variant of SYR nucleic acid/gene also can be used for the auxiliary procedure of breeding of mark.This type of procedure of breeding needs to use for example EMS mutagenesis sometimes, imports allelic variation by plant is carried out mutagenic treatment; Selectively, this program can start from the set of the allele variant in non-so-called " natural " source that produces intentionally.Carry out the evaluation of allele variant by for example PCR then.Carry out the selection of the good allele variant of aim sequence afterwards, described good allele variant causes the seed production that increases.Usually the growth performance of plant that comprises the different allele variants (for example different allele variants of any among SEQ ID NO:1, SEQ ID NO:27 to SEQ ID NO:32, SEQ ID NO:36 to SEQID NO:42 and the SEQ ID NO:44) of aim sequence by monitoring is selected.Can be in the greenhouse or field monitoring growth performance.Other optional step comprise and will wherein identify plant and other plant hybridization of good allele variant.This can be used for for example producing the combination of significant phenotypic characteristic.

SYR nucleic acid or its variant also can be used as the mark that gene (described SYR nucleic acid or its variant are the parts of described gene) is carried out the heredity and the probe of physical mapping and conduct and the proterties of these gene linkages.This information can be used for plant breeding has the phenotype of expectation with generation strain.It is the nucleotide sequence of at least 15 Nucleotide that this purposes of SYR nucleic acid or its variant only needs length.SYR nucleic acid or its variant can be used as restriction fragment length polymorphism (RFLP) mark.Available SYR nucleic acid or its variant are surveyed the Southern trace (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A LaboratoryManual) of the plant genome DNA of restricted enzymic digestion.The program that uses a computer then for example MapMaker people (1987) Genomics 1:174-181 such as () Lander is carried out genetic analysis to make up genetic map with the banding pattern of gained.In addition, can use nuclei acid probe to comprise the Southern trace of the genomic dna of one group of individuality that restriction endonuclease handles, the parent and the offspring of the genetic cross of wherein should the individual representative of group determining.The separation of record dna polymorphism is used it for and is calculated position in the genetic map that uses this colony to obtain before of SYR nucleic acid or its variant people (1980) Am.J.Hum.Genet.32:314-331 such as () Botstein.

The generation and the application of the probe in the plant gene source that is used for genetic mapping have been described in Bernatzky and Tanksley (GENETICS 112 (4): 887-898,1986).Many publications have been described and have been used the genetic mapping that aforesaid method is learned or its modification is carried out specific cDNA clone.For example, F2 hybridization colony, backcross population, panmictic population, near isogenic line (near isogenic line) and other group of individuals can be used for mapping.These class methods are known to those skilled in the art.

Nucleic acid probe also can be used for physical mapping (that is placement of sequence on physical map; Referring to people In:Non-mammalian Genomic Analysis:A Practical Guide such as Hoheisel, Academic press 1996, pp.319-346 and the reference of wherein quoting).

In another embodiment, nucleic acid probe can be used for direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154).(number kb is to hundreds of kb although the existing method of FISH mapping helps big clone; Referring to people such as Laan (1995) Genome Res.5:13-20) use, but the raising of sensitivity can allow to use short probe to carry out the FISH mapping.

Can use nucleic acid to carry out the multiple method that is used for heredity and physical mapping based on nucleic acid amplification.Example comprises the segmental polymorphism (CAPS of allele specific amplification (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplification; People such as Sheffield (1993) Genomics16:325-332), allele-specific connects people (1988) Science241:1077-1080 such as () Landegren, Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radioactivity hybridization mapping people (1997) Nat.Genet.7:22-28 such as () Walter and Happy map (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, it is right that the sequence of nucleic acid is used to design and produce the primer that is used for amplified reaction or primer extension reaction.Such primer design is known to those skilled in the art.In the method for the genetic mapping that uses PCR-based, may must in corresponding to the zone of this nucleotide sequence, identify the dna sequence dna difference of mapping between two parents of hybridizing (mapping cross).Yet this is optional usually for drawing method.

As described in the text, method of the present invention causes producing the plant of the seed production with increase.Also growth characteristics that can these are favourable and other economic favourable proterties for example other volume increase proterties, abiotic stress resistances and other to various resistances of coercing, change various constitutional featuress (architectural feature) and/or the proterties of biological chemistry and/or physiologic character combined.

The detailed description of FG-GAP

Can regulate the proteic activity of FG-GAP by the level of regulating the FG-GAP polypeptide.Selectively, also adjustable activity when the level of FG-GAP does not change.This for example can occur in when producing mutant or selection the higher or active lower variant of specific activity changes the inwardness of polypeptide mutually with wild-type.

Ding Yi term " FG-GAP albumen or its homologue " is meant and comprises (i) N end secreting signal peptide, (ii) one or more FG-GAP structural domains, and the polypeptide of the membrane spaning domain in (iii) proteinic C end half part afterwards herein.Provided example among Fig. 6.

Signal peptide is generally used for being directed to the albumen of Secretory Pathway.The algorithm (as SignalP 3.0, people such as Bendtsen, J.Mol.Biol., 340:783-795,2004) that uses a computer is easily predicted the existence of secretion signal.Common secretion signal is made up of positively charged n-district, ensuing hydrophobicity n-district and neutrality, polarity c-district.In addition, with respect to the amino-acid residue of cleavage site on position-3 and-1 normally little with the neutral amino-acid residue.

Membrane spaning domain length is about 15 to 30 amino acid and is made up of the hydrophobic residue that forms the α spiral usually.Usually based on hydrophobicity prediction they (people such as Klein for example, Biochim.Biophys.Acta 815,468,1985; Or people such as Sonnhammer, In J.Glasgow, T.Littlejohn, F.Major, R.Lathrop, D.Sankoff and C.Sensen compile, Proceedings of the SixthInternational Conference on Intelligent Systems for Molecular Biology, the 175-182 page or leaf, Menlo Park, CA, 1998.AAAI Press.).

FG-GAP structural domain (Pfam accession number PF01839, INTERPRO entry IPR000413) is present in the integrin usually, is present in this proteinic extracellular part this its to repeat (nearly 7 copies) form.Up to the present, only at large characterized integrin from animal-origin.In SEQ ID NO:53, provided the consensus sequence of FG-GAP structural domain:

fgssvaagDlnGDGrpDlvvgaPgadggtdgsvyll，

Wherein big letter is the amino acid whose single-letter amino acid code of high conservative, and other letters are the lower amino acid whose single-letter amino acid code of conservative property.This structural domain comprises Phe-Gly-X usually _n-Gly-Ala-Pro primitive, wherein X _nThe amino acid of expression variable number.Because this consensus sequence derives from animal proteinum, itself and plant FG-GAP structural domain sequence are not exclusively mated.For example, six peptides " Pgadgg " may not be present in the plant FG-GAP structural domain.Therefore, the term " FG-GAP structural domain " that herein uses comprises SEQ ID NO:53 and has sequence to the sequence similarity of SEQ ID NO:53 at least 40% (based on using Needleman ﹠amp; Wunsch algorithm (the open point penalty in room be 10 and to extend point penalty be 0.5 in the room) the SEQ ID NO:53 that carries out and the comparison of corresponding matching sequence).

The FG-GAP structural domain also can comprise Ca ²⁺Binding site.

Preferably, FG-GAP albumen also comprises FDGYLYLI (D/E) G primitive 1 (SEQ ID NO:50).More preferably, conservative primitive 1 is FDGYLYLIDG.

Additionally and/or selectively, FG-GAP albumen can comprise one or more DGXX (D/E) primitive (conservative primitive 2, SEQ ID NO:51), and wherein X can be any amino acid.Should conservative primitive can be the part of bigger primitive DXDXDGXX (D/E) (conservative primitive 3, SEQ ID NO:52), wherein X can be any amino acid.Therefore, FG-GAP albumen preferably comprises the conservative primitive 3 of one or more copies.

Selectively, the proteic homologue of FG-GAP has the amino acid of representing with SEQ IDNO:46 50% by the preferred sequence that increases progressively, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complete sequence identity, condition is that homologous protein comprises signal peptide sequence, one or more FG-GAP structural domains, with the membrane spaning domain in proteinic C-terminal half part, and preferably also comprise conservative primitive 1, one or more in 2 or 3.Use the overall comparison algorithm for example program GAP (GCG Wisconsin Package, Accelrys) the Needleman Wunsch algorithm in (preferably using default parameter and full length protein sequence) is determined complete sequence identity.

Can use for example SMART (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Schultz, 5857-5864 of special database; People such as Letunic (2002) Nucleic Acids Res 30,242-244; ), InterPro (people such as Mulder, (2003) Nucl.Acids.Res.31,315-318; ), Prosite (Bucher and Bairoch (1994), A generalized profile syntax forbiomolecular sequences motifs and its function in automatic sequenceinterpretation. (In) ISMB-94; Proceedings 2nd International Conferenceon Intelligent Systems for Molecular Biology.Altman R., Brutlag D., KarpP., Lathrop R., Searls D., Eds., pp53-61, AAAIPress, Menlo Park; People such as Hulo, Nucl.Acids.Res.32:D134-D137, (2004)) or Pfam (people such as Bateman, Nucleic Acids Research 30 (1): 276-280 (2002)), identify the various structural domains in the FG-GAP albumen.

The method that is used to search for and identifies the FG-GAP homologue is fully in those skilled in the art's limit of power.Described method comprises uses the algorithm that is used for the comparison and the comparison of sequence well known to those skilled in the art, for example GAP (Needleman and Wunsch, J.Mol.Biol.48; 443-453 (1970)), BESTFIT (uses local homology's algorithm (Advancesin Applied Mathematics 2 of Smith and Waterman; 482-489 (1981))), BLAST (Altschul, S.F., Gish, W., Miller, W., Myers, E.W.﹠amp; Lipman, D.J., J.Mol.Biol.215:403-410 (1990)), FASTA and TFASTA (W.R.Pearson and D.J.LipmanProc.Natl.Acad.Sci.USA 85:2444-2448 (1988)), will by the sequences of SEQ ID NO:45 or 46 expressions (form that can read with computer) with can compare in the sequence that public database for example obtains in MIPS, GenBank or the EMBL nucleotide sequence database.Be used to carry out the software that BLAST analyzes and pass through the public acquisition of American National biotechnology information center (NCBI).

The proteinic example that drops under the definition of " FG-GAP polypeptide or its homologue " comprises Arabidopsis albumen (SEQ ID NO:55) and two rice albumen (SEQ ID NO:57 and 59).Also also for example proved the proteic existence of FG-GAP in fern (Ceratopteris richardii) or the orchid at the age of one hundred years old (Welwitschia mirabilis) in the neutralization of the other plant species (comprising common wheat, Zea mays, potato, aquilegia (Aquilegia sp.), colea, sweet orange (Citrus sinensis), officinalis, Populus, Euphorbia esula L (Euphorbia csula)) of lily magnolia plant door (Magnoliophyta) in the other plant taxonomical group.In table 8, provided the non-limiting example tabulation of the proteic EST of coding FG-GAP:

Table 8:

Species	The GenBank accession number	SEQ?ID?NO：
Species	The GenBank accession number	SEQ?ID?NO：	Common wheat	CK207217	16
Zea mays	AY111316	17	Common wheat	CK207217	16
Zea mays	AY111316	17	Potato	BG598275	18
Aquilegia	DT735817		Potato	BG598275	18	19
Aquilegia	DT735817		Colea	CX192752		19	20
Sweet orange	CX674859		Colea	CX192752		21	20

Officinalis	CV288972		22
Officinalis	CV288972		22	Populus	CN520999	23
Populus	CX176799	24		Populus	CN520999	23
Populus	CX176799	24	Euphorbia esula L	DV130386		25
Ceratopteris?richardii	CV736049	26	Euphorbia esula L	DV130386		25
Ceratopteris?richardii	CV736049	26	Orchid at the age of one hundred years old	DT601669		27

Protein by the genes encoding that these EST originated also is used to implement method of the present invention and falls within the scope of the invention.Those skilled in the art can use standard method to separate the complete encoding sequence of these genes.

The present invention also provides and has been selected from following isolating FG-GAP albumen in addition:

(a) by the albumen of the nucleic acid encoding of SEQ ID NO:72;

(b) albumen that comprises signal sequence, one or more FG-GAP structural domain and be positioned at the membrane spaning domain of proteinic C end half part, wherein said albumen comprises at least one among SEQ ID NO:73 to the SEQID NO:72;

(c) (a) or (b) in the active fragments of aminoacid sequence of definition, the membrane spaning domain that this active fragments comprises signal sequence, one or more FG-GAP structural domain and is positioned at proteinic C end half part.

Be appreciated that, term " FG-GAP polypeptide or its homologue " is not limited to the sequence of being represented by SEQ ID NO:46 or classifies SEQ ID NO:55,57 and 59 homologue as, but meets the demands: comprise signal peptide, one or more FG-GAP structural domain and be arranged in the membrane spaning domain of proteinic C end half part and the one or more primitives that preferably also comprise the conservative primitive of SEQ ID NO:50 to 52: or any polypeptide that has the sequence identity of the sequence at least 50% of SEQ ID NO:46 can be suitable for method of the present invention.

Plant FG-GAP albumen in the pollen development process, work (people 2001 such as Paxson-Sowders).In the dex1 mutant plant, the prototheca deposition postpones and significantly reduces.In mutant, do not exist in the generation of the normal ripple formation (rippling) and gap (spacer) of observed plasma membrane in the wild-type plant yet.FG-GAP albumen can remedy this sudden change and recover normal phenotype.

Selectively, can be by in rice, being expressed in the activity that FG-GAP albumen under the constitutive promoter control or its homologue are measured FG-GAP albumen or its homologue, this expression causes producing the plant of the seed production of comparing ground biomass with increase and/or increase with corresponding wild type plant.Can measure the increase of this seed production in several modes, for example with the form of the increase of seed gross weight, full seed number or seed overall number.

FG-GAP albumen or its homologue are by FG-GAP nucleic acid/genes encoding.Therefore the term " FG-GAP nucleic acid/gene " of definition is any nucleic acid/gene of FG-GAP albumen as defined above or its homologue of encoding herein.

The example of FG-GAP nucleic acid includes but not limited to the nucleic acid by any one expression among SEQ ID NO:45, SEQ ID NO:54, SEQ ID NO:56 or the SEQ ID NO:58.Listed the example of part FG-GAP nucleic acid in the table 8.

The present invention also provides coding FG-GAP proteic isolating nucleic acid, and described nucleic acid is selected from:

(i) nucleic acid of representing among the SEQ ID NO:72;

Proteic nucleic acid of definition in (a) to (c) above (ii) encoding;

(iii) can with the nucleotide sequence of top (i) or nucleic acid array hybridizing (ii) (preferably under stringent condition), this hybridization sequences optimized encoding albumen, described albumen comprise signal peptide, one or more FG-GAP structural domain and are arranged in the membrane spaning domain of proteic C end half part;

(iv) as (i) nucleic acid to the allele variant of (iii) nucleotide sequence;

(v) as (i) nucleic acid to the alternative splicing variant of (iii) nucleotide sequence;

(vi) Shang Mian (i) is to (part of the nucleotide sequence of any one v), this part optimized encoding albumen, described albumen comprise signal peptide, one or more FG-GAP structural domain and be arranged in the membrane spaning domain of proteic C end half part.

FG-GAP nucleic acid/gene and its variant can be suitable for implementing method of the present invention.Variant FG-GAP nucleic acid/gene comprise FG-GAP nucleic acid/gene part, allele variant, splice variant and/or can with the nucleic acid of FG-GAP nucleic acid/gene recombination.

Ding Yi term partly is meant the fragment of the DNA of coded polypeptide herein, conservative primitive one or more that described polypeptide comprises signal peptide, one or more FG-GAP structural domain and is arranged in the membrane spaning domain of proteinic C end half part and preferably also comprises SEQ ID NO:50 to 52.Preferably, this part comprises one or more conservative as defined above primitives.Can for example, FG-GAP nucleic acid prepare part by being carried out one or more disappearances.Can use part with isolating form, maybe they and other codings (or non-coding) sequence can be merged for example to produce several active albumen of combination.When merging with other encoding sequences, the gained polypeptide that the translation back produces can be bigger than the segmental size of FG-GAP of prediction.Preferably, part is the part of the nucleic acid represented by the arbitrary sequence among SEQ ID NO:45, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58 or the SEQ ID NO:72.Part can also be the part of the sequence of table 8 encoding sequence of originating.Most preferably the part of nucleic acid is shown in SEQ ID NO:45.

The another kind of variant of FG-GAP nucleic acid/gene is can be under the stringent condition that reduces, preferably under stringent condition with the nucleic acid of FG-GAP nucleic acid/gene recombination of definition above, conservative primitive one or more that this hybridization sequences coded polypeptide, described polypeptide comprise signal peptide, one or more FG-GAP structural domain and be arranged in the membrane spaning domain of proteinic C end half part and preferably also comprise SEQ ID NO:50 to 52.

Preferably, hybridization sequences be the nucleic acid that can represent with SEQ ID NO:45, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58 or SEQ ID NO:72 or with above-mentioned sequence in the sequence of part (comprising EST listed in the table 8) hybridization of any sequence.Most preferably hybridization sequences can be hybridized SEQ ID NO:45.Term " hybridization " is that title is the hybridization that defines in the part of " definition ".

FG-GAP nucleic acid or its variant can derive from any natural or artificial source.Can be from microbe-derived for example yeast or fungi or from plant, algae or animal (comprising the people) source isolating nucleic acid/gene or its variant.Can in composition and/or genome environment, modify this nucleic acid from its natural form by the manual operation of having a mind to.Nucleic acid is plant origin preferably, and it can be from identical plant species (for example identical with its plant to be imported) or can be from different plant species.Can be from dicotyledonous species, preferably from cress, more preferably from the Arabidopis thaliana isolating nucleic acid.More preferably, separate FG-GAP nucleic acid from Arabidopis thaliana, and FG-GAP nucleic acid is represented that by SEQ ID NO:45 the FG-GAP aminoacid sequence is the sequence of being represented by SEQ ID NO:46.

Can regulate the expression of nucleic acids of coding FG-GAP polypeptide or its homologue by importing genetic modification (preferably in the locus of FG-GAP gene).The locus of Ding Yi gene is used to represent genome area herein, and this zone comprises the 10kb upstream or the downstream of goal gene and coding region.

Can for example import genetic modification by any (or multiple) method of following method or by the nucleic acid that imports and express coding FG-GAP polypeptide or its homologue in plant, described method is: T-DNA activation, TILLING, directed mutagenesis, transposon mutagenesis, orthogenesis and homologous recombination.This class methods have been defined at title in the part of " definition ".After importing genetic modification, step is selected in the modified expression of the nucleic acid of just encode FG-GAP polypeptide or its homologue, and wherein the modification of this expression causes having the plant of the output of increase.

T-DNA activation, TILLING, directed mutagenesis, transposon mutagenesis and orthogenesis are the examples that can produce the technology of neomorph and FG-GAP variant.

The preferred method that is used for importing genetic modification (under this situation its needn't at the locus of FG-GAP gene) is to import and express the nucleic acid of FG-GAP polypeptide as defined above or its homologue of encoding in plant.The nucleic acid of plant to be imported can be that total length nucleic acid maybe can be part or the hybridization sequences that defines hereinbefore.Preferably, the plant of genetic modification to be imported is not the dex1 mutant plant, and the DEX1 gene does not have function (people 2001 such as Paxson-Sowders) in this mutant plant.

Defined proteinic " homologue " at title for the part of " definition ".FG-GAP polypeptide or its homologue can be the derivatives of definition in " definition " part.

FG-GAP polypeptide or its homologue can be by the alternative splicing variant codings of FG-GAP nucleic acid/gene.Term " alternative splicing variant " as defined herein.The splice variant of nucleic acid of following polypeptide of encoding is preferred, conservative primitive one or more that described polypeptide comprises signal peptide, one or more FG-GAP structural domain and is arranged in the membrane spaning domain of proteinic C end half part and preferably also comprises SEQ ID NO:50 to 52.The splice variant of the arbitrary gene in the splice variant of splice variant of representing by SEQ ID NO:45, SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO:58 or the nucleic acid represented by SEQ ID NO:72 or the gene that the sequence in the table 8 is originated more preferably.The splice variant shown in the SEQ ID NO:45 most preferably.

Homologue also can be by the allele variant of the nucleic acid of coding FG-GAP polypeptide or its homologue, the allele variant coding of the nucleic acid of optimized encoding polypeptide (one or more of conservative primitive that described polypeptide comprises signal peptide, one or more FG-GAP structural domains and is arranged in the membrane spaning domain of proteic C end half part and preferably also comprises SEQ ID NO:50 to 52).More preferably, the allele variant of coding FG-GAP polypeptide is by any expression among SEQ ID NO:45, SEQ ID NO:54, SEQ IDNO:56 or the SEQ ID NO:58.Most preferably, the allele variant of coding FG-GAP polypeptide is represented by SEQ ID NO:45.Defined allele variant in " definition " part.

According to a preferred aspect of the present invention, can expect the modulated expression of FG-GAP nucleic acid or its variant.Preferably, the modulated expression was expression.The method of cross the expressing existing lot of documents report in this area that is used for gene or gene product for example comprises the use of crossing expression, transcriptional enhancer or translational enhancer by suitable promoters driven.Can import isolating nucleic acid in the suitable position (usually in the upstream) of the polynucleotide of non-allos form, to raise the expression of FG-GAP nucleic acid or its variant as promotor or enhancer element.For example, can by change in sudden change, disappearance and/or the replacement endogenesis promoter (referring to Kmiec, United States Patent (USP) 5,565,350; People such as Zarling PCT/US93/03868), maybe can import the expression of vegetable cell with controlling gene with suitable direction and distance (with respect to gene of the present invention) with isolating promotor.The method that is used to reduce the expression of gene or gene product is also known in this area.

If expression of polypeptides expects that the 3 ' end that is desirably in the polynucleotide encoding district usually comprises polyadenylation region.Polyadenylation region can derive from natural gene, from multiple other plant gene, or from T-DNA.3 ' the end sequence that adds for example can derive from nopaline synthase or octopine synthase gene or selectively from another plant gene or more preferably from any other eukaryotic gene.

Also can add intron sequences to the encoding sequence of 5 ' non-translational region or part encoding sequence to be increased in the ripe courier's who accumulates in the cytosol amount.But being presented at the intron that comprises montage in the transcription unit of plant and animal expression construct can make genetic expression increase nearly 1000 times on mRNA and protein level, Buchman and Berg, Mol.Cell biol.8:4395-4405 (1988); People such as Callis, Genes Dev.1:1183-1200 (1987).In the time of near placing 5 of transcription unit ' end, this type of intron enhancement of genetic expression is maximum usually.The purposes of corn intron A dh1-

S introne

1,2 and 6, Bronze-1 intron is known in this area.Referring to, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994) prevailingly.

The present invention also provides the importing and/or the expression of genetic constructs and the carrier nucleotide sequence to help to be used for method of the present invention.

Therefore, the invention provides gene construct, this construct comprises:

(i) FG-GAP nucleic acid or its variant that above defines;

(iii) transcription termination sequence;

Condition be gene construct be not by people such as Hajdukiewicz (Plant Mol.Biol.25,989-994) and the pPZP type gene construct described of Paxson-Sowders (2001).

Can use recombinant DNA technology well known to those skilled in the art to make up the construct that is used for method of the present invention.Gene construct can be inserted the carrier that is suitable for being transformed into plant and is suitable for expression goal gene in transformant, the commercially available acquisition of described carrier.

Transform plant with the carrier that comprises aim sequence (that is the nucleic acid of coding FG-GAP polypeptide or its homologue).Aim sequence effectively is connected with one or more control sequences (at least with promotor).Term " controlling element ", " control sequence " and " promotor " all are used interchangeably herein, and define in the part of title for " definition ".

Advantageously, can use the expression of the promoters driven nucleotide sequence of any kind.Preferably, FG-GAP nucleic acid or its functional variant effectively are connected with constitutive promoter.Term " composing type " is as the definition of this paper.Preferably, can be preferentially in whole plants the constitutive promoter of express nucleic acid have the express spectra suitable with the GOS2 promotor.More preferably, constitutive promoter has and the identical express spectra of rice GOS2 promotor, most preferably, can be preferentially in whole plants the promotor of express nucleic acid be GOS2 promotor (Nucleotide 1 to 2193 of the sequence of representing among the SEQ ID NO:49) from rice.Should be understood that suitability of the present invention is not subject to the FG-GAP nucleic acid of being represented by SEQ ID NO:45, suitability of the present invention is not subject to the FG-GAP expression of nucleic acids by the GOS2 promoters driven yet.The example that also can be used for driving other constitutive promoters of FG-GAP expression of nucleic acid is shown in table 3 of " definition " part.

Gene construct of the present invention also can be included in keeps and/or duplicates necessary replication orgin sequence in the specific cell type.An example is to keep in bacterial cell in the gene construct with the form of additive type genetic elements (for example plasmid or clay molecule).Preferred replication orgin includes but not limited to f1-ori and colE1.

Gene construct can randomly be included in the selectable marker gene of " definition " part definition of this specification sheets.

The present invention also comprises can be by the plant of method acquisition of the present invention.Therefore the invention provides the plant that obtains by method of the present invention, this plant has imported FG-GAP nucleic acid or its variant as defined above therein.

The present invention also provides the method for the transgenic plant that are used to produce the output with increase, and this method is included in and imports and express FG-GAP nucleic acid or its variant as defined above in the plant.

More particularly, the invention provides the method for the transgenic plant that produce the output with increase, this method comprises:

(i) in plant or vegetable cell, import and express FG-GAP nucleic acid or its variant; With

(ii) culturing plants cell under the condition that promotes plant-growth and growth.

Nucleic acid directly can be imported vegetable cell or import plant itself (comprising the tissue, organ or any other part that import plant).According to preferred feature of the present invention, preferably nucleic acid is imported plant by transforming.

Term " conversion " is the defined conversion of " definition " part.

The present invention clearly extends to any vegetable cell or the plant that produces by any method described herein, with and all plant parts and propagulum.The present invention further extends to and comprises that unique requirement is genotype and/or identical genotype and/or the phenotypic characteristic of phenotypic characteristic that this offspring shows and produced by the parent in the method for the invention by the former generation conversion of any method generation in the aforesaid method or the offspring of cells transfected, tissue, organ or whole plants.The present invention also comprises the host cell that comprises isolating FG-GAP nucleic acid or its variant.Preferred host cell of the present invention is a vegetable cell.The present invention also extends to the part gathered in the crops of plant such as but not limited to seed, leaf, fruit, flower, stem culture, rhizome, stem tuber and bulb.The invention still further relates to and derive from, preferably be directed to the product of the part gathered in the crops of this type of plant, for example dried granulated feed or dry powder, oil, fat and lipid acid, starch and protein.

The present invention also comprises the purposes of FG-GAP nucleic acid or its variant and the purposes of FG-GAP polypeptide or its homologue.

Such purposes relates to the growth characteristics of improveing plant, improves output, particularly seed production especially.Seed production can comprise: in the seed overall number of the seed gross weight of increase, the full seed number of increase and increase one or multinomial.

FG-GAP nucleic acid or its variant or FG-GAP polypeptide or its homologue can be used for the procedure of breeding, in the described procedure of breeding, identify can with the dna marker of FG-GAP gene or its variant genetic linkage.FG-GAP nucleic acid/gene or its variant, or FG-GAP polypeptide or its homologue can be used for defining molecule marker.This DNA or protein labeling can be used for the procedure of breeding has the output of increase with selection plant then.FG-GAP gene or its variant can for example be the genes of being originated by listed sequence in the nucleic acid of any expression among SEQ ID NO:45, SEQID NO:54, SEQ ID NO:56, SEQ ID NO:58 and the SEQ ID NO:72 or the table 8.

The allele variant of FG-GAP nucleic acid/gene also can be used for the marker-assisted breeding program.This type of procedure of breeding needs the mutagenic treatment by plant sometimes, uses for example EMS mutagenesis to import allelic variation; Selectively, this program can start from the set of the allele variant in non-what is called " natural " source that produces intentionally.For example carry out the evaluation of allele variant then by PCR.Carry out to cause the selection of good allele variant of the aim sequence of the output that increases afterwards.Usually select by the growth performance of monitoring following plant, described plant comprises the different allele variants of aim sequence, for example the different allele variants of a sequence in the encoding sequence that listed sequence is originated in any of SEQ ID NO:45, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58 and SEQ ID NO:72 or the table 8.Can be in the greenhouse or field monitoring growth performance.Other optional step comprise and will wherein identify plant and other plant hybridization of good allele variant.This can be used for for example producing the combination of significant phenotypic characteristic.

FG-GAP nucleic acid or its variant also can be used as gene (described FG-GAP nucleic acid or its variant are the parts of described gene) are carried out the heredity and the probe of physical mapping and is used as mark with the proterties of these gene linkages.This information can be used for plant breeding has the phenotype of expectation with generation strain.It is the nucleotide sequence of at least 15 Nucleotide that this purposes of FG-GAP nucleic acid or its variant only needs length.FG-GAP nucleic acid or its variant can be used as restriction fragment length polymorphism (RFLP) mark.Available FG-GAP nucleic acid or its variant are surveyed the Southern trace (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, ALaboratory Manual) of the plant genome DNA of restrictive diges-tion.The program that uses a computer then for example MapMaker people (1987) Genomics 1:174-181 such as () Lander is carried out genetic analysis to make up genetic map with the banding pattern of gained.In addition, can use nuclei acid probe to comprise the Southern trace of the genomic dna of one group of individuality that restriction endonuclease handles, the parent and the offspring of the genetic cross of wherein should the individual representative of group determining.The separation of record dna polymorphism is used it for and is calculated position in the genetic map that uses this colony to obtain before of FG-GAP nucleic acid or its variant people (1980) Am.J.Hum.Genet.32:314-331 such as () Botstein.

The generation and the application of the probe in the plant gene source that is used for genetic mapping have been described in Bernatzky and Tanksley (Plant Mol.Biol.Reporter 4:37-41,1986).Many publications have been described the genetic mapping that uses aforesaid method or its modification that specific cDNA clone is carried out.For example, F2 hybridization colony, backcross population, panmictic population, near isogenic line and other group of individuals can be used for mapping.These class methods are known to those skilled in the art.

Can use nucleic acid to carry out the multiple method that is used for heredity and physical mapping based on nucleic acid amplification.Example comprises the polymorphism (CAPS of allele specific amplification (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplified fragment; People such as Sheffield (1993) Genomics16:325-332), allele-specific connects people (1988) Science241:1077-1080 such as () Landegren, Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radioactivity hybridization mapping people (1997) Nat.Genet.7:22-28 such as () Walter and Happy map (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, it is right that the sequence of nucleic acid is used to design and produce the primer that is used for amplified reaction or primer extension reaction.This type of primer design is known to those skilled in the art.In the method for the genetic mapping that uses PCR-based, may must in corresponding to the zone of this nucleotide sequence, identify the dna sequence dna difference of mapping between the parent of hybridizing.Yet this is optional usually for drawing method.

As noted before, method of the present invention causes producing the plant of the output with increase.Also growth characteristics that can these are favourable and other economic favourable proterties for example other increase output proterties, to different resistances of coercing, change various constitutional featuress (architectural feature) and/or the proterties of biological chemistry and/or physiologic character combined.

The detailed description of CYP90B

Ding Yi term " CYP90B polypeptide or its homologue " is meant and comprises following polypeptide herein: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes to take place in any position.

In addition, CYP90B polypeptide or its homologue can also comprise (i) and have SEQ ID NO:78 is surpassed the sequence of 50% identity and (ii) steroid 22-α hydroxylase enzymatic activity.

In the table 9a of this specification sheets, provided the above example of the CYP90B polypeptide of definition.

CYP90B polypeptide or its homologue are by CYP90B nucleic acid/genes encoding.Therefore the term " CYP90B nucleic acid/gene " of definition is encode the CYP90B polypeptide of above definition or any nucleic acid/gene of its homologue herein.

In the CYP superfamily protein, be included in the CYP90B polypeptide of the present invention, the various structural domains that exist are known in this area, and can use for example SMART (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Schultz, 5857-5864 of general database; People such as Letunic (2002) Nucleic Acids Res 30,242-244; Http:// smart.embl-heidelberg.de/), InterPro (people such as Mulder, (2003) Nucl.Acids.Res.31,315-318; Http:// www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profilesyntax for biomolecular sequences motifs and its function in automaticsequence interpretation, in ISMB-94; The 2nd international conference record Altman R. of molecular biology intelligence system, Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAI Press, Menlo Park; People such as Hulo, Nucl.Acids.Res.32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (people such as Bateman, NucleicAcids Research 30 (1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/) identify.

For Arabidopis thaliana also can http://arabidopsis-P450.biotec.uiuc.edu/cgi-bin/p450.pl or more commonly on CYP homepage http://drnelson.utmem.edu/CytochromeP450.html. the search special database.The typical structure territory of finding among the CYP can be by Kalb ﹠amp; Four structural domains of the initial A to D that describes of Loper ((1988) Proc Natl Acad Sci 85:7221-7225).A structural domain (being also referred to as spiral I) comprises consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser, and thinks and combine with dioxygen.The B structural domain is the steroid binding domains.The D structural domain is corresponding to the protoheme binding domains and comprise the most distinctive CYP amino acid consensus sequences (Phe-X-X-Gly-X-Arg-X-Cys-X-Gly) (Figure 10 and 13).

Can use the sequence alignment method that is used for comparison as mentioned above to identify the existence of consensus sequence.In some cases, can adjust default parameter to change the severity of search.For example be used to report the statistical significance threshold value at the coupling of database sequence (being called " expectation " value), to show the lower coupling of severity by using BLAST, can increasing.By this method, can identify short coupling almost completely.To fully recognize as those skilled in the art, can identify herein consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (comprising consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser as defined above) in the A structural domain of CYP90B polypeptide of definition in this mode.

At CYP P450 albumen, another structural domain of identifying in the CYP90B polypeptide particularly of the present invention can be albumen n end be used for the film target, be rich in hydrophobic residue for example the anchor structure territory of Leu, Ile, Val, Phe and Ala (anchor domain).N end anchor structure length of field is generally 20 to 40 amino acid, but can shorter (down to 10 amino acid) or longer (going up to 100 amino acid).Pass through translocation domain, N end anchor structure territory separates with proteic rest part (globular domain), described translocation domain comprised (at least 2 of cluster alkaline residues before proline(Pro) bunch, Lys or Arg, be called halt-transfer signal), it forms hinge between above-mentioned anchor structure territory and proteic globular domain.The typical consensus sequence of translocation domain is Lys/Arg-Lys/Arg-(X) _3-9-Pro-Pro-Gly (Figure 10 and 13).Can identify this consensus sequence as mentioned above.

Can identify easily that N holds the existence in hydrophobic anchor structure territory.Can use software program from the ExPASy server, particularly ProtParam instrument people (2003) ExPASy:theproteomics server for in-depth protein knowledge and analysis.NucleicAcids Res 31:3784-3788 such as () Gasteiger E first order calculation amino acid is formed (representing with %), whether is rich in specific amino acid to determine the polypeptide structure territory.The composition and the average amino acid in the Swiss-Prot protein sequence database of target protein can be formed (representing with %) relatively then.In this database, the adding and be 34.04% of the mean value of Leu (L), Ile (I), Val (V), Phe (F) and Ala (A).For example, the N of SEQ ID NO:78 holds hydrophobic anchor structure territory to comprise these identical hydrophobic residues of 62.5%.As defined here, N hold hydrophobic anchor structure territory have with the Swiss-Prot protein sequence database in proteinic average amino acid form (representing) and compare higher hydrophobic amino acid content (representing) with % with %.

Specific software is ProtScale (people (2005) ProteinIdentification and Analysis Tools on the ExPASy Server.In John M.Walker such as Gasteiger for example, ed:The Proteomics Protocols Handbook, Humana Press pp.571-607) can calculate and be presented on the spectrum that produces by any amino acid scale (aminoacid scale) on the selected protein.The amino acid scale is determined by giving all kinds of amino acid whose numerical value.The scale of normal use is hydrophobicity or wetting ability scale and secondary structure conformation parameter scale.One of hydrophobic amino acid scale of the most normal use is by Kyte ﹠amp; Doolittle ((1982) J.Mol.Biol.157:105-132) produces, and wherein gives the hydrophobic amino acid positive number, gives the hydrophilic amino acid negative.For example, for the hydrophobicity of CYP90B polypeptide of the present invention, the ProtScale output spectra clearly illustrates that approximately preceding 34 N terminal amino acids are hydrophobic domains, because these amino acid are positioned at 0 top (Figure 12) that demarcates.This zone is corresponding to N end anchor structure territory.Those skilled in the art will very clear this analysis.

The routine techniques of knowing in this area be can use, for example, CYP90B polypeptide or its homologue easily identified by sequence alignment.The sequence alignment method that is used to compare is known in this area, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses the algorithm ((1970) J Mol Biol48:443-453) of Needleman and Wunsch, with the comparison of finding to make the number maximization of coupling and making minimized two complete sequences of number in room.BLAST algorithm people (1990) J Mol Biol 215:403-10 such as () Altschul calculates per-cent sequence identity and carries out the statistical analysis of two similaritys between the sequence.Being used to carry out the software that BLAST analyzes can obtain publicly by American National biotechnology information center.Can use for example can be at the ClustalW multisequencing alignment algorithm (version 1.83) of http://clustalw.genome.jp/sit-bin/nph-ClustalW acquisition, the methods of marking that uses default paired comparison parameter and represent with per-cent easily identifies to comprise to have the CYP90B homologue that SEQ ID NO:78 is surpassed the sequence of 50% identity.Can carry out a small amount of human-edited to optimize the comparison between the conservative primitive, this it will be apparent to those skilled in the art that.

Table 9a provides the example of CYP90B polypeptide or its homologue (by the coding of the polynucleotide sequence accession number in the bracket).Table 9b provides the portion C YP90B sequence of encoding part CYP90B open reading-frame (ORF) (ORF).

Table 9:a) example of CYP90B homologue

Title	NCBI or TIGR Nucleotide accession number	Nucleotide SEQ ID NO	The polypeptide SEQ ID NO of translation	The insertion state	The source
Title	NCBI or TIGR Nucleotide accession number	Nucleotide SEQ ID NO	The polypeptide SEQ ID NO of translation	The insertion state	The source	Orysa_CYP90B	AB206579.1	77	78	Total length ORF	Rice
Arath_CYP90Bl	NM_114926.2	79	80	Total length ORF	Arabidopis thaliana	Orysa_CYP90B	AB206579.1	77	78	Total length ORF	Rice
Arath_CYP90Bl	NM_114926.2	79	80	Total length ORF	Arabidopis thaliana	Sacof_CYP90B＊＊	CA092707.1CF574030.1CA217329.1	81	82	Total length ORF	Sugarcane (saccharum officinarum)
Allce_CYP90B	TC2113		83	84	Total length ORF	Sacof_CYP90B＊＊	CA092707.1CF574030.1CA217329.1	81	82	Total length ORF	Sugarcane (saccharum officinarum)	Onion (Allium cepa)
Allce_CYP90B	TC2113		83	84	Total length ORF	Zinel_CYP90B	AB231155		85	86	Total length ORF	Onion (Allium cepa)	Youth-and-old-age (Zimniaelegans)

Medtr_CYP90B ^*	AC147964.10	87	88	Total length ORF	Puncture vine clover (Medicago trunculata)
Medtr_CYP90B ^*	AC147964.10	87	88	Total length ORF	Puncture vine clover (Medicago trunculata)	Poptr_CYP90B¨＊＊	CK090847.1CV280598.1DT503533.1	89	90	Total length ORF	Comospore poplar (Populus trichocarpa)

Table 9:b) has the example of the CYP90B of part open reading-frame (ORF) (ORF)

Title	NCBI or TIGR Nucleotide accession number	Nucleotide SEQ ID NO	The polypeptide SEQ ID NO of translation	The insertion state	The source
Title	NCBI or TIGR Nucleotide accession number	Nucleotide SEQ ID NO	The polypeptide SEQ ID NO of translation	The insertion state	The source	Aqufo_CYP90B ^＊＊	DR940523.1 DR940522.1	91	92	Part of O RF	Aquilegia?Formosa　x?Aquilegia?pu?bescens
Triae_CYP90B 5 ' end	BQ620306.1	93	94	Part of O RF	Common wheat	Aqufo_CYP90B ^＊＊	DR940523.1 DR940522.1	91	92	Part of O RF	Aquilegia?Formosa　x?Aquilegia?pu?bescens
Triae_CYP90B 5 ' end	BQ620306.1	93	94	Part of O RF	Common wheat	Triae_CYP90B 3 ' end ^＊＊	BQ619714.1 CA715360.1	95	96	Part of O RF	Common wheat
Eupes_CYP90B	DV141872.1	97	98	Part of O RF	The serum setose thistle	Triae_CYP90B 3 ' end ^＊＊	BQ619714.1 CA715360.1	95	96	Part of O RF	Common wheat
Eupes_CYP90B	DV141872.1	97	98	Part of O RF	The serum setose thistle	Goshi_CYP90B5 ' end ^＊＊?	CO125422 DT568185.1	99	100	Part of O RF	Upland cotton
Lyces_CYP90B5 ' end ^＊＊	BF050501?AW221826.1 BM409833?	101	102	Part of O RF	Tomato	Goshi_CYP90B5 ' end ^＊＊?	CO125422 DT568185.1	99	100	Part of O RF	Upland cotton
Lyces_CYP90B5 ' end ^＊＊	BF050501?AW221826.1 BM409833?	101	102	Part of O RF	Tomato	Soltu_CYP90B 5 ' end ^＊＊?	BQ045917BQ114367	103	104	Part of O RF	Potato
Soltu_CYP90B 3 ' end ^＊＊?	BQ114368	105	106	Part of O RF	Potato	Soltu_CYP90B 5 ' end ^＊＊?	BQ045917BQ114367	103	104	Part of O RF	Potato

^*The artificial montage of carrying out from genomic clone

^*Contig (contig) from several EST accession number (showing main EST accession number) compilation; The EST sequencing quality is lower usually, can expect the minority replacement nucleic acid.

Should understand, drop on sequence under the definition of " CYP90B polypeptide or its homologue " and be not limited to the sequence represented by SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQID NO:86, SEQ ID NO:88 or SEQ ID NO:90, but comprise: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) any polypeptide of the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that allows in the A structural domain in an amino acid variation of any position generation all can be suitable for carrying out the present invention.

Dropping on sequence under the definition of " CYP90B polypeptide or its homologue " can also comprise (i) and have SEQ ID NO:78 is surpassed the sequence of 50% identity and (ii) steroid 22-α hydroxylase enzymatic activity.

CYP90B polypeptide or its homologue have 22-α hydroxylase enzymatic activity, can use the plant that has sudden change in DWF4, determine this hydroxylase activity by complementary assay.In rice (Tos2091 mutant), this type of mutant plant has been described by people such as Tanaka (US2004/0060079) in Arabidopis thaliana (dwf4 mutant) neutralization by people such as Choe people such as ((1998) Plant Cell 10:231-243).The size of these mutant plants is than the little several times of size of their corresponding wild types, that is, mutant plant is super the dwarfing.In being suitable for the recombinant DNA carrier of Plant Transformation, isolated polypeptide placed and to express under the control of promotor of this polypeptide plant.Use the technology of knowing in this area to transform mutant plant then with this carrier.If plant transformed is no longer showed the super phenotype of downgrading, show that then isolated polypeptide can show 22-α hydroxylase enzymatic activity.Such polypeptide can be suitable for implementing method of the present invention.

The example of CYP90B nucleic acid includes but not limited to the nucleic acid by any expression among SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 and the SEQ ID NO:89.CYP90B nucleic acid/gene and its variant can be suitable for implementing method of the present invention.The variant of CYP90B nucleic acid/gene comprise CYP90B nucleic acid/gene part and/or can with the nucleic acid of CYP90B nucleic acid/gene recombination.

Ding Yi term " part " is meant the fragment of the DNA of coded polypeptide herein, and described polypeptide comprises following: (a) CYP P450 structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes to take place in any position.Can for example, CYP90B nucleic acid prepare part by being produced one or more disappearances.Can use part maybe they and other codings (or non-coding) sequence can be merged for example to produce several active albumen of combination with isolating form.When merging to other encoding sequences, the polypeptide that produces after the translation of gained can be bigger than the size of partly predicting at this CYP90B.Preferably, part is the part by the nucleic acid of any expression among SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 and the SEQ ID NO:89.Most preferably part is the part of the nucleic acid represented by SEQ ID NO:77.

The another kind of variant of CYP90B nucleic acid/gene is under the stringent condition that reduces, preferably under stringent condition can with the nucleic acid of the CYP90B nucleic acid/gene recombination of definition above, this hybridization sequences coding comprises following: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow to take place the polypeptide of the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser of an amino acid variation in any position.Preferably, hybridization sequences be can with by the nucleic acid of any expression among SEQID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 and the SEQ ID NO:89 or with the above-mentioned sequence of above definition in the sequence of part hybridization of arbitrary sequence.Most preferably hybridization sequences be can with the sequence of the nucleic acid hybridization of representing by SEQ ID NO:77.Term " hybridization " is the hybridization of " definition " part definition in this specification sheets.

CYP90B nucleic acid or its variant can derive from any natural or artificial source.Can be from microbe-derived for example yeast or fungi or from plant, algae or animal (comprising the people) source isolating nucleic acid/gene or its variant.Can in composition and/or genome environment, modify this nucleic acid by the manual operation of having a mind to from its natural form.This nucleic acid is plant origin preferably, perhaps from identical plant species (for example identical with its plant to be imported) or can be from different plant species.This nucleic acid can separate from the monocotyledons species, and preferable separation is more preferably separated from Oryza (Oryza) from Gramineae, more preferably separates from rice.More preferably, shown in SEQ ID NO:77, and the CYP90B aminoacid sequence is shown in SEQ ID NO:78 from the isolating CYP90B nucleic acid of rice.

The present invention also provides isolating CYP90B albumen, and it is selected from:

(a) by the protein of the nucleic acid encoding of SEQ ID NO:117;

(b) comprise: (i) CYP structural domain A to D; (ii) N holds hydrophobic anchor structure territory; (iii) translocation domain; (iv) in the A structural domain, permission produces the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes in any position, and has protein with the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQID NO:118,99% identity by the preferred sequence that increases progressively.

The present invention also provides coding CYP90B proteic isolating nucleic acid, and it is selected from:

(i) nucleic acid represented of SEQ ID NO:117;

(ii) coding top (a) and (b) the proteic nucleic acid of middle definition;

(iii) has the nucleic acid at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% represented with SEQ ID NO:117,99% or the nucleic acid of bigger identity by the preferred sequence that increases progressively;

(iv) can be under stringent condition and top (i) nucleotide sequence to (iii) nucleic acid array hybridizing, this hybridization sequences proteins encoded, described albumen comprise (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes takes place in permission in any position, and has and the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ ID NO:118,99% or bigger identity by the preferred sequence that increases progressively;

(v) as the allele variant of (i) nucleotide sequence extremely (iv) or the nucleic acid of splice variant;

((i) is to (part of the nucleotide sequence of v) each, this part proteins encoded, described albumen comprise (i) CYP structural domain A to D vi); (ii) N holds hydrophobic anchor structure territory; (iii) translocation domain; (iv) in the A structural domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes takes place in any position in permission, and has and the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ IDNO:118,99% identity by the preferred sequence that increases progressively.

Can be by importing genetic modification (preferably in the locus of CYP90B gene), non-composing type ground increases the expression of nucleic acids of coding CYP90B polypeptide or its homologue.The locus of Ding Yi gene is used to represent genome area herein, and this genome area comprises the 10kb upstream or the downstream of goal gene and coding region.

Can for example pass through: any (or multiple) method or import genetic modification in T-DNA activation, TILLING, directed mutagenesis, orthogenesis and the homologous recombination by the nucleic acid that in plant, imports and express coding CYP90B polypeptide or its homologue.Defined aforesaid method in " definition " part.After importing genetic modification, the non-constitutive expression of the increase of the nucleic acid of just encode CYP90B polypeptide or its homologue is selected step, and wherein the increase of this non-constitutive expression causes having the plant of the output of increase.

T-DNA activation, TILLING, directed mutagenesis and orthogenesis are the examples that makes it possible to produce the technology of neomorph and CYP90B variant.

The preferred method that is used for importing genetic modification (under this situation its needn't at the locus of CYP90B gene) is the nucleic acid that imports and express coding CYP90B polypeptide or its homologue in plant.CYP90B polypeptide or its homologue are defined as comprising: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow to produce the polypeptide of the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser of an amino acid variation in any position.The nucleic acid of plant to be imported can be total length nucleic acid maybe can be as defined above the part or hybridization sequences.In addition, the nucleic acid of coding CYP90B polypeptide or its homologue can also comprise (i) and has SEQ ID NO:78 is surpassed the sequence of 50% identity and (ii) steroid 22-α hydroxylase enzymatic activity.

" definition " part at this specification sheets has defined proteinic " homologue ".CYP90B polypeptide or its homologue can be the derivatives of " definition " part definition.

CYP90B polypeptide or its homologue can be by the alternative splicing variant codings of CYP90B nucleic acid/gene.Defined term " alternative splicing variant " in " definition " part.Preferred splice variant is the splice variant of nucleic acid encoding, and described polypeptide comprises following: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes to take place in any position.In addition, CYP90B polypeptide or its homologue can also comprise (i) and have SEQ ID NO:78 is surpassed the sequence of 50% identity and (ii) steroid 22-α hydroxylase enzymatic activity.The splice variant of the nucleic acid of being represented by SEQ ID NO:77, SEQID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87 and SEQ ID NO:89 is preferred.The splice variant of the nucleotide sequence of being represented by SEQ IDNO:77 is most preferred.

Homologue also can be by the allele variant of the nucleic acid of coding CYP90B polypeptide or its homologue, the allele variant coding of the nucleic acid of the following polypeptide of particularly encoding, and described polypeptide comprises following: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes to take place in any position.In addition, CYP90B polypeptide or its homologue can also comprise (i) and have SEQ ID NO:78 is surpassed the sequence of 50% identity and (ii) steroid 22-α hydroxylase enzymatic activity.The allele variant of the nucleotide sequence of being represented by SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 and SEQ ID NO:89 is preferred.The allele variant of the nucleotide sequence of being represented by SEQ ID NO:77 is most preferred.Also defined allele variant in " definition " part.

According to a preferred aspect of the present invention, can expect the non-constitutive expression of the increase of CYP90 nucleic acid or its variant.The method that is used to increase the expression of gene or gene product is known in this area, and it for example comprises the use of crossing expression, transcriptional enhancer or translational enhancer by suitable promoters driven.Can import isolating nucleic acid as promotor or enhancer element at the correct position (usually in the upstream) of the polynucleotide of non-allos form to raise the expression of CYP90B nucleic acid or its variant.For example, can by the sudden change, the disappearance and/or the displacement change in vivo endogenesis promoter (referring to, Kmiec, United States Patent (USP) 5,565,350; People such as Zarling PCT/US93/03868), or can import the expression of vegetable cell with controlling gene with isolating promotor with suitable direction and distance (with respect to gene of the present invention).The method that is used to reduce the expression of gene or gene product is known in this area.

If the expectation expression of polypeptides, the 3 ' end that is desirably in the polynucleotide encoding district usually comprises polyadenylation region.Polyadenylation region can derive from natural gene, derives from multiple other plant gene or derives from T-DNA.3 ' the end sequence that is added into can derive from for example nopaline synthase or octopine synthase gene, or selectively derives from the other plant gene, or does not more preferably derive from any other eukaryotic gene.

Also can in the encoding sequence of 5 ' non-translational region or part encoding sequence, add intron sequences to be increased in the ripe courier's who accumulates in the cytosol amount.But be presented at and comprised the montage intron in the transcription unit of plant and animal expression construct and can make expression of gene on mRNA and protein level, increase nearly 1000 times of (Buchman and Berg (1988) Mol.Cell biol.8:4395-4405; People such as Callis (1987) Genes Dev.1:1183-1200).In the time of near placing 5 of transcription unit ' end, this intron enhancement of genetic expression is maximum usually.The purposes of corn intron A dh1-

S introne

1,2 and 6, Bronze-1 intron is known in this area.Usually referring to, TheMaize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

The present invention also provides the importing of the nucleotide sequence that helps to be used for method of the present invention and/or the genetic constructs and the carrier of expression.

Therefore, provide gene construct, it comprises:

(i) CYP30B nucleic acid or its variant that above defines;

(ii) can drive one or more control sequences of non-constitutive expression of the nucleotide sequence of (i); Randomly

(iii) transcription termination sequence.

Can use recombinant DNA technology well known to those skilled in the art to make up the construct that is used for method of the present invention.Gene construct can be inserted the carrier that is suitable for being transformed into plant and is suitable for expression goal gene in transformant, the commercially available acquisition of this carrier.Therefore the invention provides gene construct purposes in the method for the invention as defined above.

The carrier that use comprises aim sequence (that is the nucleic acid of coding CYP90B polypeptide or its homologue) transforms plant.Aim sequence effectively is connected with one or more control sequences (at least with promotor).Term " controlling element ", " control sequence " and " promotor " all are used interchangeably herein, and define in " definition " part.

Advantageously, can use any non-constitutive promoter to drive the expression of nucleotide sequence.Non-group of type promotor can be inducible promoter, i.e. response is grown, chemistry, environment or physical stimulation and have the promotor of the transcription initiation of inductive or increase.The example of inducible promoter is a stress induced promoter, the promotor that promptly is activated when plant is exposed to various stress conditions.Non-constitutive promoter can be to organize preference type promotor, promptly can be preferentially for example organizes initial promotor of transcribing in leaf, root, the seed tissue etc. at some.Be merely able to some the tissue in initial promotor of transcribing be called " tissue-specific " herein.

The method according to this invention effectively is connected CYP90B nucleic acid or its variant with non-constitutive promoter.Non-constitutive promoter only has transcriptional activity and is not to express ubiquitously in some periods of plant-growth and growth.Non-constitutive promoter can for example be seed specific promoters or root-specific promoter.Seed specific promoters can be endosperm-specific and/or embryo/aleuron specificity promoter, promptly has transcriptional activity respectively in seed endosperm and/or seed embryo and aleuron.Endosperm specificity promoter is the seed storage protein promotor preferably, more preferably endosperm specificity promoter is the prolamine promotor, more preferably endosperm specificity promoter is a rice RP6 prolamine promotor, more preferably endosperm specificity promoter is by representing that to the similar basically nucleotide sequence of SEQ ID NO:109 most preferably endosperm specificity promoter is the promotor of being represented by SEQ ID NO:109.Embryo/aleuron specificity promoter is the seed storage protein promotor preferably, more preferably embryo/aleuron specificity promoter is the oleosin promotor, more preferably embryo/aleuron specificity promoter is a rice oleosin 18kDa promotor, more preferably embryo/aleuron specificity promoter represents that by similar to SEQ ID NO:110 basically nucleotide sequence most preferably embryo/aleuron specificity promoter is represented by SEQ ID NO:110.Root-specific promoter is the Rcc3 promotor preferably, and root-specific promoter is rice Rcc3 promotor (people (1995) Plant Mol Biol 27 (2) such as Xu: 237-48) preferably.

Should be understood that suitability of the present invention is not subject to the CYP90B nucleic acid of being represented by SEQ ID NO:77, suitability of the present invention is not subject to the CYP90B expression of nucleic acids by RP6 prolamine or 18kDa oleosin promoters driven yet.The example that also can be used for implementing other non-constitutive promoters of the inventive method is shown in table 4 of " definition " part.

Opposite with above-mentioned promotor, constitutive promoter has transcriptional activity and expression ubiquitously in plant basically in most of periods of plant-growth and growth.This type of constitutive promoter will be not used in the method for the present invention of implementing.The example of this type of promotor also sees " definition " partly (referring to table 3).

Randomly, one or more terminator sequences also can be used in the construct of plant to be imported.Defined term " terminator " in " definition " part.

Gene construct of the present invention also can comprise in order to keep and/or duplicate necessary replication orgin sequence in specific cell type.An example is when gene construct need be kept in bacterial cell with additive type genetic elements (for example plasmid or clay molecule).Preferred replication orgin includes but not limited to f1-ori or colE1.

Gene construct can randomly comprise the selectable marker gene of " definition " part definition.

In preferred embodiments, provide gene construct, this construct comprises:

(i) CYP90B nucleic acid or its variant as defined above;

(ii) can drive the promotor of the non-constitutive expression of nucleotide sequence of (i); Randomly

(iii) transcription termination sequence.

Non-constitutive promoter is seed specific promoters preferably.Seed specific promoters can be endosperm-specific and/or embryo/aleuron specificity promoter, promptly has transcriptional activity respectively in seed endosperm and/or seed embryo and aleuron.Endosperm specificity promoter is the seed storage protein promotor preferably, more preferably endosperm specificity promoter is the prolamine promotor, more preferably endosperm specificity promoter is a rice RP6 prolamine promotor, more preferably endosperm specificity promoter is by representing that to the similar basically nucleotide sequence of SEQ ID NO:109 most preferably endosperm specificity promoter is the promotor of being represented by SEQ ID NO:109.Embryo/aleuron specificity promoter is the seed storage protein promotor preferably, more preferably embryo/aleuron specificity promoter is the oleosin promotor, more preferably embryo/aleuron specificity promoter is a rice oleosin 18kDa promotor, more preferably embryo/aleuron specificity promoter is represented by similar to SEQID NO:110 basically nucleotide sequence, and most preferably embryo/aleuron specificity promoter is the promotor of being represented by SEQID NO:110.The present invention also provides the above construct purposes in the method for the invention of definition.

The present invention also comprises can be by the plant of method acquisition of the present invention.Therefore the invention provides can be by plant, its plant part or the vegetable cell of method acquisition of the present invention, and described plant or its part or cell comprise transgenosis CYP90B nucleic acid or its variant.

The present invention also provides and has been used to produce the method for comparing the transgenic plant of the output with increase with suitable control plant, and described method is included in the plant and imports and non-constitutive expression CYP90B nucleic acid or its variant.

More preferably, the invention provides the method for the transgenic plant that are used to produce the output with increase, described method comprises:

(i) in plant, plant part or vegetable cell, import and non-constitutive expression CYP90B nucleic acid or its variant; With

Nucleic acid directly can be imported vegetable cell or import plant itself (comprising the tissue, organ or any other part that import plant).According to a preferred aspect of the present invention, preferably nucleic acid is imported plant by transforming.

Term " conversion " is the conversion of definition in " definition " part.

The present invention clearly extends to any vegetable cell or the plant that produces by any method described herein, extends to its all plant parts and its propagulum.The present invention extends further to and comprises that by the former generation conversion of any aforesaid method generation or the offspring of cells transfected, tissue, organ or complete plant, unique requirement is genotype and/or identical genotype and/or the phenotypic characteristic of phenotypic characteristic that this offspring shows and produced by the parent in the method for the invention.

The present invention also comprises the isolating CYP90B nucleic acid that comprises non-constitutive expression or the host cell of its variant.Preferred host cell of the present invention is a vegetable cell.

The present invention also extends to the part gathered in the crops of plant, such as but not limited to seed, leaf, fruit, flower, stem, rhizome, stem tuber and bulb.The invention still further relates to the product of the part gathered in the crops that is directed to such plant, for example dried particle is raised grain or dry powder, oil, fat and lipid acid, starch or protein.

The present invention also comprises the purposes of CYP90B nucleic acid or its variant and the purposes of CYP90B polypeptide or its homologue.This purposes relates to increases plant biomass as defined above in the method for the invention.

CYP90B nucleic acid or its variant, or CYP90B polypeptide or its homologue can be used for the procedure of breeding, in the described procedure of breeding, identify can with the dna marker of CYP90B gene or its variant genetic linkage.CYP90B nucleic acid/gene or its variant, or CYP90B polypeptide or its homologue can be used for defining molecule marker.This DNA or protein labeling can be used for the procedure of breeding to select to have the plant of the output that increases as defined above in the method for the invention then.CYP90B gene or its variant can for example be the nucleic acid by any expression among SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 and the SEQ ID NO:89.

The allele variant of CYP90B nucleic acid/gene also can be used for the marker-assisted breeding program.This type of procedure of breeding needs sometimes by plant is carried out mutagenic treatment, and for example using, EMS mutagenesis imports allelic variation; Selectively, this program can start from the set of the allele variant in non-so-called " natural " source that produces intentionally.Carry out the evaluation of allele variant by for example PCR then.Select step afterwards, can cause good allele variant output, aim sequence that increases with selection.Usually the growth performance of plant that comprises the different allele variants (for example different allele variants of any among SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87 and the SEQ ID NO:89) of aim sequence by monitoring is selected.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises and will wherein identify plant and other plant hybridization of good allele variant.This can be used for for example producing the combination of significant phenotypic characteristic.

CYP90B nucleic acid or its variant also can be used as the mark that gene is carried out the heredity and the probe of physical mapping and conduct and the proterties of these gene linkages, and wherein CYP90B nucleic acid or its variant are the parts of described gene.This information can be used for plant breeding has the phenotype of expectation with generation strain.It is the nucleotide sequence of at least 15 Nucleotide that this purposes of CYP90B nucleic acid or its variant only needs length.CYP90B nucleic acid or its variant can be used as restriction fragment length polymorphism (RFLP) mark.Available CYP90B nucleic acid or its variant are surveyed the Southern trace (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, ALaboratory Manual) of the plant genome DNA of restrictive diges-tion.The program that uses a computer then for example MapMaker people (1987) Genomics 1:174-181 such as () Lander is carried out genetic analysis to make up genetic map with the banding pattern of gained.In addition, can use nuclei acid probe to comprise the Southern trace of the genomic dna of one group of individuality that restriction endonuclease handles, the parental generation and the offspring of the genetic cross of wherein should the individual representative of group determining.The separation of record dna polymorphism is used it for and is calculated position in the genetic map that uses this colony to obtain before of CYP90B nucleic acid or its variant people (1980) Am.J.Hum.Genet.32:314-331 such as () Botstein.

(GENETICS 112 (4): described the generation and the application of the probe in the plant gene source that is used for genetic mapping 887-898) at Bernatzky and Tanksley (1986).Many publications have been described the genetic mapping that uses aforesaid method or its modification that specific cDNA clone is carried out.For example, the individuality of F2 hybridization colony, backcross population, panmictic population, near isogenic line and other groups can be used for mapping.These class methods are known to those skilled in the art.

Method of the present invention causes producing the plant of the output with increase as noted before.Also can with the output of this increase and other economic favourable proterties for example other increase output proterties, to other abiotic and biological resistances of coercing, modify different structure feature and/or biological chemistry and/or physiological characteristic proterties combined.

The detailed description of CDC27

The CDC27 polypeptide in this area be know and can be by conservative NH ₂(at least one TPR structural domain is at NH for end regions (referring to Figure 16) and at least 5 TPR structural domains ₂In the end regions) existence easily identify.In addition, the CDC27 polypeptide can also comprise and has the sequence that SEQ ID NO:130 is surpassed 30% identity.

In yeast and higher eucaryote, in mitotic division (comprising APC protein ingredient CDC16, CDC23 and CDC27), transcribe, the input of montage, protein and neural (Goebl and the Yanagida1991 of taking place, Trends Biochem Sci 16, there is the TPR primitive in the multiple protein that works in 173-177).The minimum consensus sequence of the suggestion of TPR primitive is: X ₃-W-X ₂-L-G-X ₂-Y-X ₈-A-X ₃-F-X ₂-A-X ₄-P-X ₂, wherein any amino acid of X=(people 1994 such as Lamb, EMBO J 13,4321-4328).Total residue can be showed significant degeneracy, and non-total residue is showed minimum homology or do not had homology.The hydrophobicity of important total residue seemingly and size but not its identity.In natural CDC27 albumen, TPR forms αLuo Xuanjiegou; Its tandem repetitive sequence be organized into the superhelix that is ideally suited as the interface of protein identification (Groves and Barford 1999, Curr Opin Struct Biol 9,383-389).In the α spiral, there are two amphipathic structural domains usually, one is positioned at NH ₂End regions, (people 1990 such as Sikorski, Cell 60,307-317) near the COOH end regions for another.In addition, single TPR primitive also can be dispersed in the whole protein sequence.

The natural CDC27 of total length comprises at least 5 TPR usually, preferred 6 TPR, and more preferably 7 TPR, the major part of these TPR is positioned at the COOH end regions.As shown in Figure 16, usually at the NH of natural CDC27 polypeptide ₂There is 1 TPR structural domain in end regions, can be created in NH although can exist maybe ₂End regions comprises the variant CDC27 sequence of a plurality of TPR.

Any CDC27 polypeptide all can be by making the NH of polypeptide ₂At least one TPR structural domain inactivation in the end regions, and can be used in method of the present invention.The method that is used for inactivation in this area be know and comprise: amino acid whose elimination or displacement, under this situation, NH ₂The amino acid whose elimination or the displacement of at least one TPR structural domain in the end regions; Or mutating technology, but for example with L-Ala displacement conserved amino acid or with the amino acid (for example Serine, Threonine or tyrosine) of amino-acid substitution phosphorylation that can not phosphorylation or vice versa (depending on that phosphorylated protein is active or non-activity); Or be used for any other method of inactivation.

For the application's purpose, with the proteic NH of CDC27 ₂End regions is considered as total length CDC27 sequence (from NH ₂Hold to COOH end) the first two/one (referring to Figure 16); Preferably with the proteic NH of CDC27 ₂End regions is considered as total length CDC27 sequence (from NH ₂Hold to COOH end) first three/one; With another preferred feature according to the present invention, the proteic N end regions of CDC27 is considered as total length CDC27 sequence (from NH ₂Hold to COOH end) preceding 166 amino acid.

At NH ₂The example of CDC27 polypeptide that end regions has the TPR structural domain of at least one inactivation is the polypeptide of being represented by SEQ ID NO:130, and its nucleic acid sequence encoding is represented by SEQ ID NO:129.

Following table 10 has provided some examples of CDC27 sequence; Can be by for example using the NH of any method for deactivating discussed above with polypeptide ₂At least one TPR structural domain inactivation in the end regions makes these sequences can be used for method of the present invention.

The example of table 10:CDC27 polypeptide

Title	NCBI Nucleotide accession number	Nucleotide SEQ IDNO	The polypeptide SEQ ID NO of translation	The source
Title	NCBI Nucleotide accession number	Nucleotide SEQ IDNO	The polypeptide SEQ ID NO of translation	The source	CDC27B	AC006081
	129	130	Arabidopis thaliana		CDC27B	AC006081
	129	130	Arabidopis thaliana	CDC27B/Hobbit	AJ487669		131	132	Arabidopis thaliana
CDC27a	NM_112503.2\|	133	134	CDC27B/Hobbit	AJ487669		131	132	Arabidopis thaliana	Arabidopis thaliana
CDC27a	NM_112503.2\|	133	134	CDC27	AP003539.3	135	136	Rice		Arabidopis thaliana
CDC27	BG887406.1 ^＊BG590616.1 DN939130.1 CV470643.1	137	139	CDC27	AP003539.3	135	136	Rice	Potato
CDC27	BG887406.1 ^＊BG590616.1 DN939130.1 CV470643.1	137	139	CDC27/nuc2+	NM_001020032.1	139	140	Schizosaccharomyces pombe (Schizosaccharomyces pombe)	Potato
CDC27/BimA	X59269.1	141	142	CDC27/nuc2+	NM_001020032.1	139	140	Schizosaccharomyces pombe (Schizosaccharomyces pombe)	Aspergillus niger (Aspergillusniger)
CDC27/BimA	X59269.1	141	142	CDC27	NM_001256.2	143	144	Human (Homo sapiens)	Aspergillus niger (Aspergillusniger)
CDC27?5’	CA102186.1 ^＊CA279358.1	145	146	CDC27	NM_001256.2	143	144	Human (Homo sapiens)	Sugarcane

CDC27?3’

CA197669.1 ^＊CA197670.1CA203636.1CA232307.1

147

148

Sugarcane

^*Contig (showing main EST accession number) from several EST accession number compilations; The EST sequencing quality is lower usually, can expect the minority replacement nucleic acid.

Only provide the sequence of describing in the table 10 with illustrational form.Other examples of coding total length or part of polypeptide (can use it to obtain full length sequence by using ordinary method) are provided in Figure 19.The NH that should be understood that at polypeptide ₂Have any CDC27 peptide sequence of TPR structural domain of at least one inactivation or the nucleic acid/gene of such polypeptide of encoding in the end regions and all can be suitable for implementing method of the present invention.

Can use the routine techniques of knowing in this area for example easily to identify other CDC27 polypeptide by sequence alignment.Subsequently can be for example by using any method for deactivating discussed above, with the NH of polypeptide ₂At least one TPR structural domain inactivation in the end regions makes the sequence that is identified can be used for method of the present invention.The sequence alignment method that is used to compare is known in this area, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses the comparison of algorithm ((1970) J Mol Biol 48:443-453) to find the making number maximization of coupling and to make minimized two complete sequences of number in room of Needleman and Wunsch.BLAST algorithm people (1990) J Mol Biol 215:403-10 such as () Altschul calculates per-cent sequence identity and carries out the statistical analysis of two similaritys between the sequence.Can obtain to be used to carry out the software that BLAST analyzes publicly by American National biotechnology information center.Can use for example can be at the ClustalW multiple sequence alignment algorithm (version 1.83) of http://clustalw.genome.jp/sit-bin/nph-ClustalW acquisition, the methods of marking that uses default paired comparison parameter and represent with per-cent is easily identified the homologue of CDC27.Can carry out a small amount of human-edited to optimize the comparison between the conservative primitive, this it will be apparent to those skilled in the art that.

Can use for example SMART (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Schultz, 5857-5864 of special database; People such as Letunic (2002) Nucleic Acids Res 30,242-244; Http:// smart.embl-heidelberg.de/), InterPro (people such as Mulder, (2003) Nucl.Acids.Res.31,315-318; Http:// www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecularsequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; The 2nd international conference of molecular biology intelligence system record Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAIPress, Menlo Park; People such as Hulo, Nucl.Acids.Res.32:D134-D137, (2004), http://www.expasy.org/prosite/), Pfam (people such as Bateman, Nucleic Acids Research 30 (1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/) or ProDom (Servant F, Bru C, Carrere S, Courcelle E, Gouzy J, Peyruc D, Kahn D (2002) ProDom:Automated clustering of homologous domains.Briefings in Bioinformatics. the 3rd volume, no 3:246-251), the various structural domains in the evaluation CDC27 albumen are the TPR structural domain for example.

The sequence of mentioning among table 10 and Figure 19 can be thought the homologue of CDC27 polypeptide." definition " part at this specification sheets has defined proteinic " homologue ".Preferred homologue is to have and the total length CDC27 albumen of being represented by SEQ ID NO:132 at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or the aminoacid sequence of bigger sequence identity by the preferred sequence that increases progressively.

Can be by for example using any method for deactivating discussed above, with the NH of polypeptide ₂At least one TPR structural domain inactivation in the end regions makes homologue, lineal homologue and collateral line homologue can be used for method of the present invention.

By the yeast two-hybrid analysis in vivo with by co-immunoprecipitation at external (people (1994) EMBO J 13 (18): 4321-4328 such as Lam; Ollendorf ﹠amp; Donoghue (1997) J BiolChem 272 (51): 32011-32018), shown that two other PROTEIN C DC16 of people and yeast CDC27 polypeptide and APC mixture and CDC23 interact.Such interaction can be used for identifying the CDC27 polypeptide, so that by for example using the NH of any method for deactivating discussed above with described polypeptide ₂At least one TPR structural domain inactivation in the end regions and make it can be used in method of the present invention.

NH at polypeptide ₂The CDC27 polypeptide of TPR structural domain that has at least one inactivation in the end regions is by so-called modified CDC27 nucleic acid/genes encoding.Therefore, the term " modified CDC27 nucleic acid/gene " of definition is the NH that is coded in polypeptide herein ₂Any nucleic acid/the gene of CDC27 polypeptide that has the TPR structural domain of at least one inactivation in the end regions.

CDC27 nucleic acid or modified CDC27 nucleic acid/gene can derive from any natural or artificial source.Can be from microbe-derived, for example yeast or fungi or from plant, algae or animal-origin isolating nucleic acid/gene.Can in composition and/or genome environment, modify this nucleic acid from its natural form by the manual operation of having a mind to.Nucleic acid is plant origin preferably, and it can be from identical plant species (for example identical with its plant to be imported) or can be from different plant species.Can be from dicotyledonous species, preferably from Cruciferae, more preferably from the Arabidopis thaliana isolating nucleic acid.More preferably, represent by SEQ ID NO:129 from the isolating modified CDC27 nucleic acid of Arabidopis thaliana, at amino acid whose NH ₂The CDC27 that has the TPR of at least one inactivation in the end regions is the polypeptide of being represented by SEQ ID NO:130.

CDC27 nucleic acid/gene can be can be under the stringent condition that reduces, preferably under stringent condition with nucleic acid by the CDC27 nucleic acid/gene recombination of any expression among SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137 or the SEQ ID NO:141.Most preferably hybridization sequences is can be to the sequence of the nucleic acid hybridization represented by SEQ ID NO:129 or SEQ IDNO:131.This hybridization sequences can be by for example using the NH of any method for deactivating discussed above with its encoded polypeptide ₂At least one TPR structural domain inactivation in the end regions and can be used for method of the present invention.

Term " hybridization " is the hybridization of this specification sheets " definition " part definition.

CDC27 nucleic acid or modified CDC27 nucleic acid/gene can be the form of alternative splicing variant.Defined the alternative splicing variant in " definition " part.The splice variant of the arbitrary sequence in the above-mentioned CDC27 nucleotide sequence (being SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ IDNO:135, SEQ ID NO:137 or SEQ ID NO:141) is preferred.The splice variant of the nucleotide sequence of being represented by SEQ ID NO:129 or SEQ ID NO:131 is most preferred.Can be by for example using the NH of any method for deactivating discussed above with the CDC27 polypeptide of coding ₂At least one TPR structural domain inactivation in the end regions, and make this type of splice variant can be used for method of the present invention.

CDC27 nucleic acid or modified CDC27 nucleic acid/gene can be the allele variant forms of the nucleic acid of the CDC27 polypeptide (the TPR structural domain that comprises at least one inactivation at the NH2 end regions) that blocks of coding.The allele variant of the nucleotide sequence of being represented by SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137 or SEQ ID NO:141 is preferred.The allele variant of the nucleotide sequence of being represented by SEQ ID NO:129 or SEQ ID NO:131 is most preferred.The natural existence of allele variant, these natural allelic application are included within the method for the present invention.Allele variant comprises single nucleotide polymorphism (SNP) and little insertion/deletion polymorphism (INDEL).The size of INDEL is usually less than 100bp.SNP and INDEL form one group of maximum sequence variants in the natural polymorphism strain of most of biologies.Can be by for example using the NH of any method for deactivating discussed above with the CDC27 polypeptide of coding ₂At least one TPR structural domain inactivation in the end regions makes this equipotential genetic mutation can be used for method of the present invention.

Can produce CDC27 nucleic acid or modified CDC27 nucleic acid/gene by directed mutagenesis.Can obtain several method and realize directed mutagenesis, the most frequently used method is based on the method for PCR (Current Protocols in Molecular Biology, Wiley Eds Http:// www.4ulr.com/ Products/currentprotocols/index.html).

Also can produce CDC27 nucleic acid or modified CDC27 nucleic acid/gene by orthogenesis (about detailed content more referring to " definition " part).

Can be by for example using the NH of any method for deactivating discussed above with the CDC27 polypeptide of coding ₂At least one TPR structural domain inactivation in the end regions makes the variant that produces by directed mutagenesis or orthogenesis can be used for method of the present invention.

Can increase the modified CDC27 nucleic acid/expression of gene of the following CDC27 polypeptide of coding by importing genetic modification (preferably in the locus of CDC27 gene), described CDC27 polypeptide is at the NH of polypeptide ₂End regions has the TPR structural domain of at least one inactivation.The locus of Ding Yi gene is used to represent genome area herein, and this zone comprises the 10KB upstream or the downstream of goal gene and coding region.

Preferably import genetic modification by the nucleic acid that imports and express the following CDC27 polypeptide of coding in plant, described CDC27 polypeptide is at the NH of polypeptide ₂End regions has the TPR structural domain of at least one inactivation.After importing genetic modification, just be coded in the NH of polypeptide ₂The modified expression of nucleic acids of CDC27 polypeptide that end regions has the TPR structural domain of at least one inactivation increases (in stem end meristematic tissue), the selection step of choosing wantonly, and the increase of described expression causes having the plant of the output of increase.

According to a preferred aspect of the present invention, can expect the expression of the increase of CDC27 nucleic acid or its variant.The method that is used to increase the expression of gene or gene product is known in this area, and it for example comprises the use of crossing expression, transcriptional enhancer or translational enhancer by suitable promoters driven.Can import isolating nucleic acid as promotor or enhancer element at the correct position (usually in the upstream) of the polynucleotide of non-allos form to raise the CDC27 expression of nucleic acids.For example, can by the sudden change, the disappearance and/or the displacement change in vivo endogenesis promoter (referring to, Kmiec, United States Patent (USP) 5,565,350; People such as Zarling PCT/US93/03868), or can import the expression of vegetable cell with controlling gene with isolating promotor with suitable direction and distance (with respect to gene of the present invention).

If the expectation expression of polypeptides, the 3 ' end that is desirably in the polynucleotide encoding district usually comprises polyadenylation region.Polyadenylation region can derive from natural gene, derives from multiple other plant gene or derives from T-DNA.3 ' end sequence to be added can derive from for example nopaline synthase or octopine synthase gene, or selectively derives from the other plant gene, or does not more preferably derive from any other eukaryotic gene.

Also can in the encoding sequence of 5 ' non-translational region or part encoding sequence, add intron sequences to be increased in the ripe courier's who accumulates in the cytosol amount.But be presented at and comprised the montage intron in the transcription unit of expression construct of plant and animal and can make expression of gene on mRNA and protein level, increase nearly 1000 times of (Buchman and Berg (1988) Mol.Cell biol.8:4395-4405; People such as Callis (1987) Genes Dev.1:1183-1200).In the time of near placing 5 of transcription unit ' end, this intron enhancement of genetic expression is maximum usually.The purposes of corn intron A dh1-

S introne

Therefore, provide gene construct, it comprises:

(i) be coded in the NH of polypeptide ₂End regions has the CDC27 nucleic acid of CDC27 polypeptide of the TPR structural domain of at least one inactivation;

(ii) can preferentially in stem end meristematic tissue, drive one or more control sequences of the nucleotide sequence expression of (i); Randomly

(iii) transcription termination sequence.

Can use recombinant DNA technology well known to those skilled in the art to make up this type of gene construct.Gene construct can be inserted the carrier that is suitable for being transformed into plant and is suitable for expression goal gene in transformant, the commercially available acquisition of this carrier.Therefore the invention provides the above gene construct purposes in the method for the invention of definition.

Use comprises aim sequence and (that is, is coded in the NH of polypeptide ₂End regions has the nucleic acid of CDC27 polypeptide of the TPR structural domain of at least one inactivation) carrier transform plant.Aim sequence effectively is connected with one or more control sequences (at least with promotor) that can preferentially drive expression in axis end meristematic tissue.Term " controlling element ", " control sequence " and " promotor " all are used interchangeably herein, and define in the part of " definition ".

The NH of polypeptide will be coded in ₂End regions has CDC27 nucleic acid or the variant and the stem end meristematic tissue promotor of CDC27 polypeptide of the TPR structural domain of at least one inactivation, preferably effectively is connected with early stage stem end meristematic tissue promotor." the early stage stem end meristematic tissue promotor " of definition herein is the promotor that has transcriptional activity in embryo globular stage to the seedling phase in stem end meristematic tissue, and know these periods to those skilled in the art.That mentions preferentially increase to express in stem end meristematic tissue herein, mean mainly in stem end meristematic tissue, to increase to express, and except owing to seepage promotor former thereby any residual expression that causes, get rid of the expression at the elsewhere of plant.Preferably, early stage stem end meristematic tissue promotor is that the OSH1 promotor is (from rice; SEQ ID NO:151 (people such as Matsuoka, (1993) Plant Cell 5:1039-1048; People such as Sato, (1996) ProcNatl Acad Sci U S A 93 (15): 8117-22).Should be understood that suitability of the present invention is not subject to the modified CDC27 nucleic acid of being represented by SEQ ID NO:129, suitability of the present invention is not subject to the modified CDC27 expression of nucleic acids by the OSH1 promoters driven yet.The example of other early stage stem end meristematic tissue promotors is shown in the table 5 of " definition " part.These are the members from collateral line homology or lineal homogenic KNOX family 1 class homoeosis frame.Be to be understood that following tabulation is non-exhaustive.

The construct that randomly, also one or more terminator sequences can be used for plant to be imported.Defined term " terminator " in " definition " part in this manual.

Gene construct of the present invention also can comprise in order to keep and/or duplicate necessary replication orgin sequence in specific cell type.An example is need be in bacterial cell in the form maintenance gene construct with additive type genetic elements (for example plasmid or clay molecule).Preferred replication orgin includes but not limited to f1-ori and colE1.

The present invention also comprises can be by the plant of method acquisition of the present invention.Therefore the invention provides and can comprise vegetable cell by plant or its part of method acquisition of the present invention, described plant or plant part comprise the NH that is coded in polypeptide ₂End regions has the CDC27 nucleic acid of CDC27 polypeptide of the TPR structural domain of at least one inactivation, and wherein this nucleic acid effectively is connected with stem end meristematic tissue promotor.

The present invention also provides and has been used to produce the method for comparing the transgenic plant of the number seeds with increase with suitable control plant, and this method is included in and imports and express the NH that is coded in polypeptide in the plant ₂End regions has the CDC27 nucleic acid of CDC27 polypeptide of the TPR structural domain of at least one inactivation, and this CDC27 nucleic acid is under the control of stem end meristematic tissue promotor.

More particularly, the invention provides and be used to produce the method for comparing the transgenic plant of the number seeds with increase with suitable control plant, this method comprises:

(i) the CDC27 nucleic acid of importing and expression coding CDC27 polypeptide in plant, plant part or vegetable cell, described CDC27 polypeptide is at the NH of polypeptide ₂End regions has the TPR structural domain of at least one inactivation, and described nucleic acid is under the control of stem end meristematic tissue promotor; With

Defined term " conversion " in " definition " part.

The present invention clearly extends to any vegetable cell or the plant that produces by any method described herein and extends to its all plant parts and its propagulum.The present invention extends further to and comprises that the former generation conversion that produces by any method in the aforesaid method or the offspring of cells transfected, tissue, organ or complete plant, unique requirement are that this offspring shows genotype and/or identical genotype and/or the phenotypic characteristic of phenotypic characteristic with parent's generation in the method for the invention.

The present invention also comprises the host cell of the isolating CDC27 nucleic acid that comprises coding CDC27 polypeptide, and described CDC27 polypeptide is at the NH of polypeptide ₂End regions has the TPR structural domain of at least one inactivation, and this nucleic acid is under the control of stem end meristematic tissue promotor.Preferred host cell of the present invention is a vegetable cell.

The part gathered in the crops that the present invention also extends to plant for example but is not limited to seed, leaf, fruit, flower, stem, rhizome, stem tuber and bulb.The invention still further relates to and derive from, preferably be directed to the product of the part gathered in the crops of such plant, for example dried granulated feed or powder, oil, fat and lipid acid, starch or protein.

The present invention also comprises the purposes of the isolating CDC27 nucleic acid of coding CDC27 polypeptide, and described CDC27 polypeptide is at the NH of polypeptide ₂End regions has the TPR structural domain of at least one inactivation, and described nucleic acid is under the control of stem end meristematic tissue promotor.This purposes relates to increases plant biomass as defined above in the method for the invention.

The enforcement of the inventive method causes producing the plant of comparing the number seeds with increase with suitable control plant.Also can be with increase and other economic favourable proterties of this number seeds, for example other increase output proterties, to other abiotic and biological resistances of coercing, modify various constitutional featuress and/or biological chemistry and/or physiological characteristic proterties combined.

The detailed description of AT hook

AT hook structure territory is in the polypeptide of knowing and be present in the transcription factor family that belongs to relevant with Chromatin Remodeling usually in this area.AT hook primitive is made up of about about 13 (about sometimes 9) amino acid, these amino acid participate in DNA in conjunction with and have a preference to the zone of being rich in A/T.In Arabidopsis, there are at least 34 albumen that comprise AT hook structure territory.These albumen are along the total homology of most of sequence, and wherein AT hook structure territory is special high conservative region.In Figure 23 and following table 11 illustrated AT hook structure territories; Also referring to the suitable note of SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169 and SEQ ID NO:171 (wherein the position in AT hook structure territory spells out).As shown in the comparison of Figure 23, some variations in the AT hook structure territory allow.Usually, one or two AT hook structure territory is positioned at before the DUF296 structural domain.The AT hook structure territory of herein mentioning is meant by the preferred sequence that increases progressively to have peptide sequence with the identity of the AT hook structure territory at least 70%, 75%, 80%, 85%, 90% of SEQ ID NO:153 or 95%, for convenience's sake, the AT hook structure territory of this SEQ ID NO:153 is repeated in this: RRPRGRPAGSKNK (the AT hook structure territory of SEQ ID NO:153).

DUF296 structural domain (being called IPR005175 in Interpro) is also known in this area.At Figure 23 and following table 11 illustrated DUF296 structural domains; Also referring to the suitable note of SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169 and SEQ ID NO:171, wherein the position of DUF296 structural domain is spelt out.As shown in the comparison of Figure 23, the variation in the DUF296 structural domain is admissible, and the while, it still can easily be accredited as the DUF296 structural domain owing to the existence of the amino-acid residue of some high conservatives.Usually, there are one or two AT hook structure territories before the DUF296 structural domain.

According to a preferred aspect of the present invention, the polypeptide that comprises AT hook structure territory and DUF296 structural domain comprises 1 primitive in the following primitive extraly:

Primitive 1 (SEQ ID NO:190): QGQ V/I GG; Or

Primitive 2 (SEQ ID NO:191): ILSLSGSFLPPPAPP; Or

Primitive 3 (SEQ ID NO:192): NATYERLP; Or

Primitive 4 (SEQ ID NO:193): have 0 or 1 SFTNVAYERLPL that changes at the amino acid of any position; Or

Primitive 5 (SEQ ID NO:194): have 0,1 or 2 GRFEILSLTGSFLPGPAPPGSTGLTIYLAGGQGQVVGGSVVG that changes at the amino acid of any position.

According to preferred feature of the present invention, the sequence that is suitable for method of the present invention is to comprise AT hook structure territory (defined as mentioned) and DUF296 structural domain (defined as mentioned) and the polypeptide of primitive 2 (defined as mentioned) or the nucleic acid of this polypeptide of encoding.

Be appreciated that, sequence of describing in the table 1 and the sequence that shows in the comparison of Figure 23 just are used for the example of the sequence of method of the present invention, have any polypeptide of AT hook structure territory and DUF296 structural domain or any nucleic acid of coding said polypeptide and all can be suitable for implementing method of the present invention.

Table 11: comprise example and the sequence of these structural domains and the description of their corresponding position of the aminoacid sequence of AT hook structure territory and DUF296 structural domain

	SEQ?ID?NO	Species	Position, AT hook structure territory	Sequence A T hook structure territory	Duf296 structural domain position	Sequence D UF296 structural domain
	SEQ?ID?NO	Species	Position, AT hook structure territory	Sequence A T hook structure territory	Duf296 structural domain position	Sequence D UF296 structural domain	1	SEQIDNO：153 CDS3129	ORYSA	97-109	rrprgrpagsknk	124-241	lrthvmevaggcdisesittfarrrqrgvcvlsgagtvtnvtlrqpasqgavvalhgrfe?ilslsgsflpppappeatgl tvylaggqgqvvggsvvgal?taagpvvimaasfanavy?
2	SEQIDNO：155 CDS3128	ORYSA	97-109	RRPRGRPPGSKNK	109-227	lrahilevgsgcdvfecvst?yarrrqrgvcvlsgsgvvtnvtlrqpsapagavvslhgrf?eilslsgsflpppappgats ltiflaggqgqvvggnvvga?lyaagpviviaasfanvay?	1	SEQIDNO：153 CDS3129	ORYSA	97-109	rrprgrpagsknk	124-241
2	SEQIDNO：155 CDS3128	ORYSA	97-109	RRPRGRPPGSKNK	109-227		3	SEQIDNO：157 LOTCO- Ap006863.1 (gi68264919)	LOTUS	81-93	rrprgrpagsknk	108-225	lkthvmevadgcdivdsvsn?farrrqrgvcimsgtgtvtnvtlrqpassgavvtlhgrfe ilslagsflpppappaasgl tiylaggqgqvvggsvvgal iasgpvvimaasfsnaay
4	SEQIDNO：159 NP_192942	ARATH	119-131	RRpRGRPAGSKNK	145-263	lrthvmeigdgcdivdcmat?farrrqrgvcvmsgtgsvtnvtirqpgsppgsvvslhgrf?eilslsgsflpppappaatg lsvylaggqgqvvggsvvgp llcsgpvvvmaasfsnaay	3	SEQIDNO：157 LOTCO- Ap006863.1 (gi68264919)	LOTUS	81-93	rrprgrpagsknk	108-225
4	SEQIDNO：159 NP_192942	ARATH	119-131	RRpRGRPAGSKNK	145-263		5	SEQIDNO：161 NP_194012	ARATH	105-117	rrprgrpagsknk	132-252	farrrqrgvcvmsgtgnvtnvtirqpgshpspgsvvslhg?rfeilslsgsflpppappta tglsvylaggqgqvvggsvv gpllcagpvvvmaasfsna
6	SEQIDNO：163 NP_182067	ARATH	89-101	rrprgrpagsknk	116-237	lkshvmevangcdvmesvtvfarrrqrgicvlsgngavtnvtirqpasvpgggssvvnlhgrfeilslsgsflpppappa asgltiylaggqgqvvggsv?vgplmasgpvvimaasfgnaay	5	SEQIDNO：161 NP_194012	ARATH	105-117	rrprgrpagsknk	132-252

7	SEQIDNO：165 At3g60870/At_NP_191646	ARATH	59-71	rrprgrpagsknk	86-201	frchvmeitnacdvmeslavfarrrqrgvcvltgngavtnvtvrqpgggvvslhgrfeil?slsgsflpppappaasglkv ylaggqgqviggsvvgplta?sspvvvmaasfgnasy
7	SEQIDNO：165 At3g60870/At_NP_191646	ARATH	59-71	rrprgrpagsknk	86-201		8	SEQIDNO：167 CDS0185	ARATH	88-100	rrprgrppgsknk	115-233	lqshvleiatgadvaeslna?farrrgrgvsvlsgsglvtnvtlrqpaasggvvslrgqfeilsmcgaflptsgspaaaagltiylagaqgqvvgggvagpliasgpviviaatfcnaty
9	SEQIDNO：169 PROT?encoded?by?AK107405	ORYSA	111-123	rrprgrpagsknk	138-256	lrahvlevasgcdlvdsvat farrrqvgvcvlsatgavtnvsvrqpgagpgavvnltgrfdilslsgsflpppappsatg ltvyvsggqgqvvggtvagpliavgpvvimaasfgnaay?	8	SEQIDNO：167 CDS0185	ARATH	88-100	rrprgrppgsknk	115-233
9	SEQIDNO：169 PROT?encoded?by?AK107405	ORYSA	111-123	rrprgrpagsknk	138-256		10	SEQIDNO：171 NP_912386.1	ORYSA	45-57	rrprgrppgsknk	72-190	mrshvleiasgadiveaiagfsrrrqrgvsvlsgsgavtnvtlrqpagtgaaavalrgrf?eilsmsgaflpapappgatg?lavylaggqgqvvggsvmge?liasgpvmviaatfgnaty?
11	SEQIDNO：173 Le_BT013387	LYCES	54-66	rrprgrpagsknk	81-198	lrahilevssghdvfesvat?yarkrqrgicilsgsgtvnnvtirqpqaagsvvtlhgrfeilslsgsflpppappgatsl tiylaggqgqvvggnvvgal?iasgpviviassftnvay?	10	SEQIDNO：171 NP_912386.1	ORYSA	45-57	rrprgrppgsknk	72-190
11	SEQIDNO：173 Le_BT013387	LYCES	54-66	rrprgrpagsknk	81-198		12	SEQIDNO：175 CDS312s	ARATH	34-46	rrprgrpagsknk	61-180	lrshvlevtsgsdiseavst yatrrgcgvciisgtgavtnvtirqpaapagggvitlhgr?fdilsltgtalpppappgag gltvylaggqgqvvggnvag sliasgpvvlmaasfanavy
13	SEQIDNO：177 CDS3399	ARATH	80-92	rrprgrpagsknk	107-232	lkshvmeiasgtdvietlat?farrrqrgicilsgngtvanvtlrqpstaavaaapggaavlalqgrfeilsltgsflpgp	12	SEQIDNO：175 CDS312s	ARATH	34-46	rrprgrpagsknk	61-180

						appgstgltiylaggqgqvvggsvvgplmaagpvmliaatfsnaty
						appgstgltiylaggqgqvvggsvvgplmaagpvmliaatfsnaty	14	SEQIDNO：179 PRO?AK110263	ORYSA	35-47	rrprgrppgsknk	62-179	lrshvmevaggadvaesiahfarrrqrgvCvlsgagtvtdvalrqpaapsavvalrgrfe?ilsltgtflpgpappgstgl tvylaggqgqvvggsvvgtl?taagpvmv
15	SEQIDNO：181 At4g14465/NP_567432	ARATH	67-79	rrprgrppgsknk	94-211	lrshvleisdgsdvadtiah?fsrrrqrgvcvlsgtgsvanvtlrqaaapggvvslqgrfe?ilsltgaflpgpsppgstgl tvylagvqgqvvggsvvgpl?laigsvmviaatfsnaty	14	SEQIDNO：179 PRO?AK110263	ORYSA	35-47	rrprgrppgsknk	62-179
15	SEQIDNO：181 At4g14465/NP_567432	ARATH	67-79	rrprgrppgsknk	94-211		16	SEQIDNO：183 CDS4145	ARATH	82-94	rrprgrppgsknk	109-226	lrahilevtngcdvfdcvat?yarrrqrgicvlsgsgtvtnvsirqpsaagavvtlqgtfeilslsgsflpppappgatsl tiflaggqgqvvggsvvgel?taagpviviaasftnvay?
17	SEQIDNO：185 XP_473716	ORYSA	82-94	rrprgrppgsknk	109-227	lrahilevgsgcdvfecvst?yarrrqrgvcvlsgsgvvtnvtlrqpsapagavvslhgrf?eilslsgsflpppappgats ltiflaggqgqvvggnvvga?lyaagpviviaasfanvay?	16	SEQIDNO：183 CDS4145	ARATH	82-94	rrprgrppgsknk	109-226
17	SEQIDNO：185 XP_473716	ORYSA	82-94	rrprgrppgsknk	109-227		18	SEQIDNO：187 NP_181070	ARATH	78-90	rrprgrpagsknk	105-222	lrahilevgsgcdvfecist yarrrqrgicvlsgtgtvtnvsirqptaagavvtlrgtfeilslsgsflpppappgatsl tiflagaqgqvvggnvvgel?maagpvmvmaasftnvay?
19	SEQIDNO：189 TC102931	TC102931	86-98	rrprgrpagsknk	113-230	lrshvmevangcdimesvtvfarrrqrgvcilsgsgtvtnvtlrqpaspgavvtlhgrfe?ilslsgsflpppappaasgl aiylaggqgqvvggsvvgpl?lasgpvvimaasfgnaay	18	SEQIDNO：187 NP_181070	ARATH	78-90	rrprgrpagsknk	105-222

Those skilled in the art can easily identify the polypeptide that comprises AT hook structure territory and DUF296 structural domain by using technology and the instrument known in this area.Can carry out this evaluation by the sequence alignment that uses GAP, BESTFIT, BLAST, FASTA and TFASTA to be used for the sequence comparison.GAP uses the comparison of algorithm ((1970) J Mol Biol 48:443-453) to find the making number maximization of coupling and to make minimized two complete sequences of number in room of Needleman and Wunsch.BLAST algorithm people (1990) J Mol Biol 215:403-10 such as () Altschul calculates per-cent sequence identity and carries out the statistical analysis of two similaritys between the sequence.Can obtain to be used to carry out the software that BLAST analyzes publicly by American National biotechnology information center.Can use for example can be at the ClustalW multisequencing alignment algorithm (version 1.83) of http://clustalw.genome.jp/sit-bin/nph-ClustalW acquisition, the methods of marking that uses default paired comparison parameter and represent with per-cent is easily identified the polypeptide that comprises AT hook structure territory and DUF296 structural domain.Can carry out a small amount of human-edited to optimize the comparison between the conservative primitive, this it will be apparent to those skilled in the art that.

The database that can use specialization is SMART (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Schultz, 5857-5864 for example; People such as Letunic (2002) Nucleic Acids Res 30,242-244; Http:// smart.embl-heidelberg.de/), InterPro (people such as Mulder, (2003) Nucl.Acids.Res.31,315-318; Http:// www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecularsequences motifs and its function in automatic sequence interpretation, ISMB-94; The 2nd international conference record Altman R. of molecular biology intelligence system, Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAI Press, Menlo Park; People such as Hulo, Nucl.Acids.Res.32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (people such as Bateman, Nucleic AcidsResearch 30 (1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/), identify AT hook structure territory and DUF296 structural domain.

Sequence of mentioning in the table 11 or the sequence of using above-mentioned technology (for example sequence alignment) to identify can think to comprise the homologue of the polypeptide of AT hook structure territory and DUF296 structural domain, described homologue also comprises AT hook structure territory and DUF structural domain, but this homologue can change at the elsewhere of sequence." definition " part has in this manual defined proteinic " homologue ".Preferred homologue is to have at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or the aminoacid sequence of bigger sequence identity by the aminoacid sequence that the preferred sequence that increases progressively is represented SEQ ID NO:153, and this homologue comprises AT hook structure territory and DUF296 structural domain and more preferably also comprises primitive 2.

Comprising the polypeptide of AT hook structure territory and DUF296 structural domain or the homologue of this polypeptide can be the derivative of " definition " part definition in this specification sheets.

Any nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain can be suitable for method of the present invention.The example of such sequence comprises the nucleotide sequence of being represented by SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168 and SEQ IDNO:170..

The variant of nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain also can be suitable for implementing method of the present invention, and condition is the polypeptide that the variant coding comprises AT hook structure territory and DUF296 structural domain.This type of nucleic acid variant can be the coding polypeptide that comprises AT hook structure territory and DUF296 structural domain nucleic acid part and/or can comprise the nucleic acid of nucleic acid hybridization of the polypeptide of AT hook structure territory and DUF296 structural domain with coding.

Can be for example the nucleic acid of polypeptide by coding being comprised AT hook structure territory and DUF296 structural domain produce one or more and lack and prepare part.Can use part maybe they and other codings (or non-coding) sequence can be merged for example to produce several active albumen of combination with isolating form.When merging to other encoding sequences, the polypeptide that produces after the translation of gained can be than big at the size of this part prediction.Preferably, part is the part by the nucleic acid of any expression among SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ IDNO:164, SEQ ID NO:166, SEQ ID NO:168 and the SEQ ID NO:170.Most preferably part is the part of the nucleic acid represented by SEQ ID NO:152, the polypeptide that this part coding comprises AT hook structure territory and DUF296 structural domain and more preferably also comprises primitive 2.

Another kind of nucleic acid variant is can be under the stringent condition that reduces, and preferably comprises the nucleic acid of nucleic acid hybridization of the polypeptide of AT hook structure territory and DUF296 structural domain under stringent condition with coding.Preferably, hybridization sequences be can with by the nucleic acid of any expression among SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168 and the SEQ ID NO:170 or with the above-mentioned sequence of above definition in the sequence of part hybridization of arbitrary sequence.Most preferably, hybridization sequences is the sequence of the nucleic acid hybridization that can represent with SEQ ID NO:152, the polypeptide that this hybridization sequences coding comprises AT hook structure territory and DUF296 structural domain and more preferably comprises primitive 2.

Term " hybridization " is as the definition in this specification sheets " definition " part.

Another kind of nucleic acid variant is the alternative splicing variant in the definition of " definition " part.The splice variant of the nucleotide sequence of being represented by SEQ IDNO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ IDNO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:168 and SEQ ID NO:170 is preferred.The splice variant of the nucleotide sequence of being represented by SEQID NO:152 is most preferred, the polypeptide that this splice variant coding comprises AT hook structure territory and DUF296 structural domain and more preferably comprises primitive 2.

Another kind of nucleic acid variant is the allele variant in the definition of " definition " part.The allele variant of the nucleotide sequence of being represented by SEQ IDNO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ IDNO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:168 and SEQ ID NO:170 is preferred.The allele variant of the nucleotide sequence of being represented by SEQ ID NO:152 is most preferred, the polypeptide that this equipotential genetic mutation coding comprises AT hook structure territory and DUF296 structural domain and more preferably comprises primitive 2.

Also can pass through orthogenesis (referring to " definition " part) and obtain the nucleic acid variant.

Also can use directed mutagenesis to produce the variant of nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain.Referring to " definition " part.

The nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain can derive from any natural or artificial source.Can be from microbe-derived for example yeast or fungi or from plant, algae or animal-origin isolating nucleic acid/gene or its variant.Can in composition and/or genome environment, modify this nucleic acid by the manual operation of having a mind to from its natural form.Nucleic acid is plant origin preferably, can be from identical plant species (for example identical with its plant species to be imported) or can be from different plant species.Can be from the dicotyledons species, preferably from the monocotyledons species for example rice separate this nucleic acid.More preferably, the coding rice nucleic acid of polypeptide that comprises AT hook structure territory and DUF296 structural domain is represented that by SEQID NO:152 the polypeptide that is encoded is the polypeptide of being represented by SEQ ID NO:153.

Can be by importing genetic modification (preferably comprising in the locus of gene of polypeptide of AT hook structure territory and DUF296 structural domain), the expression of nucleic acids of regulating coding AT hook at coding.The locus of Ding Yi gene is used to represent genome area herein, and this zone comprises the 10kb upstream or the downstream of goal gene and coding region.

Can be for example by following method: any (or multiple) method and import genetic modification in T-DNA activation, TILLING and the homologous recombination by in monocotyledons, importing and express the nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain.About the details of T-DNA activation, TILLING and homologous recombination referring to " definition " part.After importing genetic modification, just coding comprise the expression of the increase of nucleic acid in endosperm tissue of the polypeptide of AT hook structure territory and DUF296 structural domain, selects step, and the expression of this orientation causes having the plant of the seed production of increase.

The selection that is used for the promotor of T-DNA activation label under situation of the present invention can be preferentially to instruct any promotor of expressing at monocotyledonous endosperm tissue.

T-DNA activates and TILLING makes it possible to produce the example of technology of the neomorph of nucleic acid of the polypeptide that comprises AT hook structure territory and DUF296 structural domain and variant of encoding.

The preferred method that is used for importing genetic modification (it needn't comprise the locus of nucleic acid/gene of the polypeptide of AT hook structure territory and DUF296 structural domain at coding under this situation) is to import and express the nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain in plant.The nucleic acid of plant to be imported can be that total length nucleic acid maybe can be part or any other variant nucleic acid, and condition is the polypeptide that the variant nucleic acid encoding comprises AT hook structure territory and DUF296 structural domain.

Method of the present invention depends on preferentially increases the expression of nucleic acids that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain in monocotyledonous endosperm tissue.This can be used for realizing by the making of expression, transcriptional enhancer or translational enhancer of crossing by suitable promoters driven.Can import isolating nucleic acid at the correct position (usually in the upstream) of the polynucleotide of non-allos form as promotor or enhancer element, more than the gene/nucleic acid of the tone coded polypeptide that comprises AT hook structure territory and DUF296 structural domain or the expression of its variant.For example, can by the sudden change, the disappearance and/or the displacement change in vivo endogenesis promoter (referring to, Kmiec, United States Patent (USP) 5,565,350; People such as Zarling PCT/US93/03868), or can import the expression of vegetable cell with controlling gene with isolating promotor with suitable direction and distance (with respect to gene of the present invention).

If the expectation express polypeptide, the 3 ' end that is desirably in the polynucleotide encoding district usually comprises polyadenylation region.Polyadenylation region can derive from natural gene, derives from multiple other plant gene or derives from T-DNA.3 ' the end sequence that is added into can derive from for example nopaline synthase or octopine synthase gene, or selectively derives from another kind of plant gene, or does not more preferably derive from any other eukaryotic gene.

S introne

Therefore, provide gene construct, it comprises:

(i) coding comprises the nucleic acid of the polypeptide of AT hook structure territory and DUF296 structural domain;

(ii) can in monocotyledonous endosperm tissue, drive one or more control sequences of the nucleotide sequence expression of (i); Randomly

(iii) transcription termination sequence.

The construct that the present invention also provides above definition is in the purposes of the method that is used for increasing monocotyledonous seed production.

Can use recombinant DNA technology well known to those skilled in the art to make up the construct that is used for method of the present invention.Gene construct can be inserted the carrier that is suitable for being transformed into plant and is suitable for expression goal gene in transformant, the commercially available acquisition of this carrier.The present invention also provides the construct that above defines in the purposes that is used in the method for monocotyledons increase seed production.

Use comprises the aim sequence carrier transforming monocots of (that is, coding comprises the nucleic acid of the polypeptide of AT hook structure territory and DUF296 structural domain).Aim sequence effectively is connected with one or more control sequences (at least with promotor) that can preferentially increase expression in monocotyledonous endosperm tissue.Term " controlling element ", " control sequence " and " promotor " all are used interchangeably herein, and define in " definition " part.

Endosperm specificity promoter is meant preferentially any promotor of driving purposes genetic expression in endosperm tissue.That mentions preferentially increase to express in endosperm tissue herein, be meant mainly in endosperm tissue, to increase to express, and except owing to seepage promotor former thereby any residual expression that causes, get rid of the expression of elsewhere in plant.For example, the prolamine promotor shows strongly expressed in endosperm, at meristematic tissue, more particularly has leakage expression among the discriminationcentre in stem meristematic tissue and/or the meristematic tissue.

Preferably, endosperm specificity promoter is the promotor from the prolamine gene isolation, the paddy prolamine RP6 that represents by SEQ ID NO:195 (people such as Wen, (1993) Plant Physiol101 (3): the 1115-6) promotor of promotor or similar intensity and/or have the promotor of the expression pattern similar for example to paddy prolamine promotor.Can be for example by promotor be analyzed similar intensity and/or similar expression pattern to the reporter gene coupling with the function of examining report gene in the tissue of plant.Well-known reporter gene is the colorimetric GUS dyeing that β-glucuronidase and being used for manifests the β-glucuronidase activity of plant tissue.Should be understood that suitability of the present invention is not subject to the nucleic acid of being represented by SEQ ID NO:152, suitability of the present invention is not subject to by the coding AT hook structure territory of prolamine promoters driven and the expression of nucleic acids of DUF296 structural domain yet.The example that also can be used for implementing other endosperm specificity promoters of method of the present invention is shown in table 6 of " definition " part.

Gene construct can randomly comprise the selectable marker gene of this paper definition.

In preferred embodiments, provide gene construct, it comprises:

(ii) can preferentially in monocotyledonous endosperm tissue, drive the prolamine promotor of the nucleotide sequence expression of (i); Randomly

(iii) transcription termination sequence.

The present invention also comprises can be by the monocotyledons of method acquisition of the present invention.Therefore the invention provides can be by monocotyledons, its plant part (comprising vegetable cell) of method acquisition of the present invention, described plant or its part comprise the transgenosis that coding contains the polypeptide of AT hook structure territory and DUF296 structural domain, described transgenosis and endosperm specificity promoter, preferred prolamine promotor effectively connects.

The present invention also provides and has been used to produce the monocotyledonous method of transgenosis of comparing the seed production with increase with suitable control plant, described method is included in and imports and express the nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain in the monocotyledons, and wherein said expression preferentially obtains increasing in monocotyledonous endosperm tissue.

More particularly, the invention provides the monocotyledonous method of the transgenosis that is used to produce the seed production with increase, described method comprises:

(i) in monocotyledonous endosperm tissue, import and preferentially increase the expression of nucleic acids that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain therein; With

Nucleic acid directly can be imported monocotyledonous vegetable cell or import plant itself (comprising the tissue, organ or any other part that import plant).According to a preferred aspect of the present invention, preferably nucleic acid is imported plant by transforming.

" definition " part at this specification sheets has defined term " conversion ".

The present invention clearly extends to any vegetable cell or the plant that produces by any method described herein and extends to its all plant parts and its propagulum.The present invention extends further to and comprises that the former generation conversion that produces by any aforesaid method or the offspring of cells transfected, tissue, organ or complete plant, unique requirement are that this offspring shows genotype and/or identical genotype and/or the phenotypic characteristic of phenotypic characteristic with parent's generation in the method for the invention.

The present invention also comprises the host cell of the nucleic acid of the polypeptide that comprising encodes contains AT hook structure territory and DUF296 structural domain, and wherein this nucleic acid effectively is connected with endosperm specificity promoter.Preferred host cell of the present invention is a monocot plant cell.

The present invention also extends to the monocotyledonous part of gathering in the crops such as but not limited to seed, leaf, fruit, flower, stem, rhizome, stem tuber and bulb.The invention still further relates to and derive from, preferably be directed to the product of the part gathered in the crops of such plant, for example granulated feed grain or dry powder, oil, fat and lipid acid, starch or protein.

The present invention comprises that also the nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain is using method of the present invention to increase purposes in the monocotyledonous seed production.

The detailed description of DOF transcription factor

Ding Yi term " DOF transcription factor polypeptide " is meant and comprises following feature (i) and following extraly feature (ii) or any polypeptide (iii) herein:

(i) by the preferred sequence that increases progressively the DOF structural domain of being represented by SEQ ID NO:200 or SEQ ID NO:228 had at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity; With

(ii) the DOF structural domain of being represented by SEQ ID NO:200 had at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity by the preferred sequence that increases progressively; Or

(iii) primitive I: unconverted or have that one or more conservative propertys in any position change or have one, two or three KALKKPDKILP (SEQ ID NO:229) that change at the non-conservation of any position; And/or

Primitive II: unconverted or have that one or more conservative propertys in any position change or have one, two or three DDPGIKLFGKTIPF (SEQ ID NO:230) that change at the non-conservation of any position.

In addition, comprise above-mentioned feature (i) and feature polypeptide (iii) and can comprise any one in the following primitive, any two or all three primitives:

-primitive III: unconverted or have that one or more conservative propertys in any position change or have one, two or three SPTLGKHSRDE (SEQ ID NO:231) in the non-conservative variation of any position; And/or

-primitive IV: unconverted or have that one or more conservative propertys in any position change or have one, two or three LQANPAALSRSQNFQE (SEQ ID NO:232) that change at the non-conservation of any position; And/or

-primitive V: unconverted or have that one or more conservative propertys in any position change or have one, two, three, four or five KGEGCLWVPKTLRIDDPDEAAKSSIWTTLGIK (SEQ ID NO:233) that change at the non-conservation of any position.

Comprise above-mentioned feature (i) and feature preferred polypeptide (iii) and comprise primitive I and II simultaneously.

In addition, DOF transcription factor polypeptide (at least with its natural form) has dna binding activity usually and has the activation structure territory.Can use routine techniques and method easily to determine the existence of activation structure territory and dna binding activity by those skilled in the art.

SEQ ID NO:199 (by SEQ ID NO:198 coding) is to comprise the feature (i) of above definition and the example of DOF transcription factor polypeptide (ii), that is, has at least 60% sequence identity with the DOF structural domain of representing by SEQ ID NO:200 or SEQ IDNO:228; And have at least 70% sequence identity with the DOF structural domain of representing by SEQ IDNO:200.At SEQ ID NO:202 (by SEQ ID NO:201 coding), SEQ ID NO:204 (by SEQ ID NO:203 coding), SEQ ID NO:206 (by SEQ ID NO:205 coding), SEQ ID NO:208 (by SEQID NO:207 coding), SEQ ID NO:210 (by SEQ ID NO:209 coding), SEQ ID NO:212 (by SEQ ID NO:211 coding), SEQ ID NO:214 (by SEQ ID NO:213 coding), SEQ ID NO:216 (by SEQ ID NO:215 coding), SEQ ID NO:218 (by SEQID NO:217 coding), SEQ ID NO:220 (by SEQ ID NO:219 coding), the feature (i) that comprises above definition and other examples of DOF transcription factor polypeptide have (ii) been provided among the SEQ ID NO:222 (by SEQ ID NO:221 coding).

SEQ ID NO:227 (by SEQ ID NO:226 coding) is to comprise the feature (i) of above definition and (iii) (that is, have at least 60% sequence identity with the DOF structural domain of being represented by SEQ ID NO:200 or SEQ ID NO:228; Above Ding Yi primitive I and/or primitive II) the example of DOF transcription factor polypeptide.At SEQ ID NO:235 (by SEQ ID NO:234 coding), SEQ ID NO:237 (by SEQ ID NO:236 coding), SEQ ID NO:239 (by SEQ IDNO:238 coding), SEQ ID NO:241 (by SEQ ID NO:240 coding), SEQ ID NO:243 (by SEQ ID NO:242 coding), SEQ ID NO:245 (by SEQ ID NO:244 coding), SEQ ID NO:247 (by SEQ ID NO:246 coding), SEQ ID NO:249 (by SEQID NO:248 coding), SEQ ID NO:251 (by SEQ ID NO:250 coding), SEQ ID NO:253 (by SEQ ID NO:252 coding), the feature (i) that comprises above definition and other examples of DOF transcription factor polypeptide have (iii) been provided among the SEQ ID NO:255 (by SEQ ID NO:254 coding).

Other examples of being represented by SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222 are the examples of " homologue " of the DOF transcription factor polypeptide represented by SEQ ID NO:199.

Other examples of being represented by SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255 are the examples of " homologue " of the DOF transcription factor polypeptide represented by SEQ ID NO:227.

" definition " part has in this manual defined proteinic " homologue ".

DOF transcription factor polypeptide or its homologue can be derivatives.Defined " derivative " in " definition " part in this manual.

Can use for example SMART (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Schultz, 5857-5864 of special database; People such as Letunic (2002) Nucleic Acids Res 30,242-244; Http:// smart.embl-heidelberg.de/), InterPro (people such as Mulder, (2003) Nucl.Acids.Res.31,315-318; Http:// www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecularsequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; The 2nd international conference of molecular biology intelligence system record Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAIPress, Menlo Park; People such as Hulo, Nucl.Acids.Res.32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (people such as Bateman, Nucleic Acids Research 30 (1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/), identify various structural domains DOF structural domain for example in the DOF transcription factor protein.

The example of the nucleic acid of encoding D OF transcription factor polypeptide (with its homologue) comprises by SEQ IDNO:198, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ IDNO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ IDNO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ IDNO:226, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ IDNO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ IDNO:248, SEQ ID NO:250, the nucleic acid of any expression among SEQ ID NO:252 and the SEQ ID NO:254.The variant of the nucleic acid of encoding D OF transcription factor polypeptide can be suitable for method of the present invention.Suitable variant comprise encoding D OF transcription factor polypeptide nucleic acid part and/or can with the nucleic acid of the nucleic acid/gene recombination of encoding D OF transcription factor polypeptide.Other variants comprise the splice variant and the allele variant of the nucleic acid of encoding D OF transcription factor polypeptide (with its homologue).

Ding Yi term " part " is meant that coding comprises following feature (i) and following extraly feature (ii) or the fragment of the DNA of polypeptide (iii) herein:

In addition, above-mentioned feature (iii) can comprise any one in the following primitive, any two or all three primitives:

Can for example produce one or more disappearances and prepare part by nucleic acid to encoding D OF transcription factor polypeptide.Can use part maybe they and other codings (or non-coding) sequence can be merged for example to produce several active albumen of combination with isolating form.When merging to other encoding sequences, the polypeptide that produces after the translation of gained can be bigger than the size of partly predicting at this DOF transcription factor.

Coding comprises above the part of nucleic acid of the feature (i) of definition and DOF transcription factor polypeptide (ii) preferably by the part of the nucleic acid of any one expression among SEQ ID NO:198, SEQ ID NO:201, SEQ ID NO:203, SEQID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219 and the SEQID NO:221.

Coding comprises above the part of nucleic acid of the feature (i) of definition and DOF transcription factor polypeptide (iii) preferably by the part of the nucleic acid of any one expression among SEQ ID NO:226, SEQ ID NO:234, SEQ ID NO:236, SEQID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252 and the SEQID NO:254.

The another kind of variant of DOF transcription factor nucleic acid/gene is can be under the stringent condition that reduces, preferred stringent condition down with the nucleic acid of the DOF transcription factor nucleic acid/gene recombination of definition above, this hybridization sequences is encoded and is comprised following feature (i) and following extraly feature (ii) or polypeptide (iii):

Preferably, coding comprise above the hybridization sequences of the feature (i) of definition and DOF transcription factor polypeptide (ii) be can with by the sequence of the nucleic acid hybridization of any expression among SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219 and the SEQ ID NO:221.

Preferably, coding comprise above the hybridization sequences of the feature (i) of definition and DOF transcription factor polypeptide (iii) be can with by the sequence of the nucleic acid hybridization of any expression among SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252 and the SEQ ID NO:254.

DOF transcription factor polypeptide can be encoded by the alternative splicing variant.Term " alternative splicing variant " is " the alternative splicing variant " that defines in " definition " part in this specification sheets.

Preferred splice variant is that coding comprises following feature (i) and following extraly feature (ii) or the splice variant of the nucleic acid of polypeptide (iii):

Preferred splice variant that coding comprises above the nucleic acid of the feature (i) of definition and DOF transcription factor polypeptide (ii) is by the splice variant of the nucleic acid of any one expression among SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219 and the SEQ ID NO:221.

Coding comprises above the preferred splice variant of nucleic acid of the feature (i) of definition and DOF transcription factor polypeptide (iii) preferably by the splice variant of the nucleic acid of any one expression among SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252 and the SEQ ID NO:254.

DOF transcription factor polypeptide also can be encoded by allele variant, and described allelotrope body also defines in " definition " part of this specification sheets.

Preferred allele variant is that coding comprises following feature (i) and following extraly feature (ii) or the allele variant of the nucleic acid of polypeptide (iii):

Preferred allele variant that coding comprises above the nucleic acid of the feature (i) of definition and DOF transcription factor polypeptide (ii) is by the splice variant of the nucleic acid of any one expression among SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219 and the SEQ ID NO:221.

Coding comprises above the preferred allele variant of nucleic acid of the feature (i) of definition and DOF transcription factor polypeptide (iii) preferably by the part of the nucleic acid of any one expression among SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252 and the SEQ ID NO:254.

Can use the directed mutagenesis of definition in the part of this specification sheets " definition " for example, produce above other variants of the nucleic acid of the DOF transcription factor polypeptide of definition of coding.

Also can use orthogenesis (or gene reorganization) to produce the variant of the nucleic acid of encoding D OF transcription factor polypeptide.Referring to " definition " part.

DOF transcription factor polypeptide is that plant is specific.The nucleic acid of coding said polypeptide can derive from any natural or artificial source.Can in composition and/or genome environment, modify this nucleic acid by the manual operation of having a mind to from its natural form.Preferred DOF transcription factor nucleic acid or its variant are from dicotyledons, and more preferably from Cruciferae, more preferably nucleic acid is from Arabidopis thaliana.

Can increase the expression of nucleic acids of encoding D OF transcription factor polypeptide by importing genetic modification (preferably in the locus of DOF transcription factor gene).The locus of Ding Yi gene is used to represent genome area herein, and this genome area comprises the 10kb upstream or the downstream of goal gene and coding region.

Can be for example by following method: any (or multiple) method or import genetic modification in T-DNA activation, TILLING and the homologous recombination by the nucleic acid that in plant, imports and express encoding D OF transcription factor polypeptide.Definition in " definition " part of the method for T-DNA activation, TILLING and homologous recombination such as this specification sheets.After importing genetic modification, with regard to the expression of the increase of the nucleic acid of encoding D OF transcription factor polypeptide, the selection step of choosing wantonly, wherein the expression of this increase causes having the plant of the output of increase.

T-DNA activates and TILLING is the example that makes it possible to produce the technology of neomorph and DOF transcription factor variant.

The preferred method that is used for importing genetic modification (under this situation its needn't at the locus of DOF transcription factor gene) is to import and express the above nucleic acid of the DOF transcription factor polypeptide of definition of coding in plant.The nucleic acid of plant to be imported can be total length nucleic acid maybe can be as defined above the part or hybridization sequences or other nucleic acid variant.

Method of the present invention depends on the expression of increase of the nucleic acid of encoding D OF transcription factor polypeptide.Be used to increase method existing lot of documents report in this area of the expression of gene or gene product, for example comprise, use the use of crossing expression, transcriptional enhancer or translational enhancer of suitable promoters driven.Can import isolating nucleic acid at the correct position (usually in the upstream) of the polynucleotide of non-allos form as promotor or enhancer element, more than the expression of nucleic acids of tone coded DOF transcription factor polypeptide.For example, can by the sudden change, the disappearance and/or the displacement change in vivo endogenesis promoter (referring to, Kmiec, United States Patent (USP) 5,565,350; People such as Zarling PCT/US93/03868), or can import the expression of vegetable cell with controlling gene with isolating promotor with suitable direction and distance (with respect to gene of the present invention).

S introne

1,2 and 6, Bronze-1 intron is known in this area.About general information, referring to, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Therefore, provide gene construct, it comprises:

(i) coding nucleic acid or its variant of DOF transcription factor polypeptide of definition above;

(iii) transcription termination sequence.

Can use recombinant DNA technology well known to those skilled in the art to make up the construct that is used for method of the present invention.Gene construct can be inserted carrier, the commercially available acquisition of this carrier, be suitable for being transformed into plant and be suitable in transformant, expressing goal gene.Therefore the invention provides the above gene construct purposes in the method for the invention of definition.

The carrier that use comprises aim sequence (that is the nucleic acid of encoding D OF transcription factor polypeptide) transforms plant.Aim sequence effectively is connected with one or more control sequences (at least with promotor).Term " controlling element ", " control sequence " and " promotor " all are used interchangeably herein, and define in " definition " part of this specification sheets.

Advantageously, can use the expression of promotor (no matter being natural or synthetic) the driving nucleotide sequence of any kind.

According to a preferred feature of the present invention, the constitutive promoter of definition in " definition " part of DOF transcription factor nucleic acid or its variant and this specification sheets effectively is connected.Constitutive promoter is the GOS2 promotor preferably, more preferably constitutive promoter is a rice GOS2 promotor, more preferably constitutive promoter is by representing that to the similar basically nucleotide sequence of SEQ ID NO:225 most preferably constitutive promoter is the promotor of being represented by SEQ ID NO:225.Preferred constitutive promoter is used to drive expression of nucleic acids, and described nucleic acid encoding comprises the feature (i) of above definition and (ii) (that is, the DOF structural domain of being represented by SEQ IDNO:200 or SEQ ID NO:228 had at least 60% sequence identity; With the sequence identity that the DOF structural domain of being represented by SEQ ID NO:200 is had at least 70%) DOF transcription factor polypeptide.

Should be understood that suitability of the present invention is not subject to the DOF transcription factor nucleic acid of being represented by SEQ ID NO:198, suitability of the present invention is not subject to the DOF transcription factor expression of nucleic acids by the GOS2 promoters driven yet.The example that also can be used for implementing other constitutive promoters of method of the present invention is shown in table 3 of " definition " part of this specification sheets.

According to another preferred aspect of the present invention, with the nucleic acid of encoding D OF transcription factor polypeptide and seed specific promoters (promptly, mainly in seed tissue, express, but can have residual expression promoter owing to the former of seepage promoter expression thereby at other positions of plant) effectively connect.More preferably, from the coding seed storage protein gene isolation seed specific promoters, particularly endosperm specificity promoter.Most preferably, from prolamine gene isolation endosperm specificity promoter, the paddy prolamine RP6 that represents by SEQID NO:258 (people such as Wen for example, (1993) Plant Physiol101 (3): 1115-6) promotor, or have to the promotor of the similar intensity of this paddy prolamine promotor and/or have the promotor of the expression pattern similar to this paddy prolamine promotor.Can be for example by promotor be analyzed similar intensity and/or similar expression pattern to the reporter gene coupling with the function of examining report gene in the tissue of plant.Well-known reporter gene is the colorimetric GUS dyeing that β-glucuronidase and being used for manifests the β-glucuronidase activity of plant tissue.The prolamine promotor shows strongly expressed in endosperm, in meristematic tissue, more particularly show leakage expression in stem meristematic tissue and/or merismatic discrimination centre.

According to the present invention, the preferred seed specific promoters that uses, particularly endosperm specificity promoter drives coding and comprises the feature (i) of above definition and (iii) (promptly the DOF structural domain of being represented by SEQ ID NO:200 or SEQID NO:228 is had at least 60% sequence identity; With primitive I and primitive II) the expression of nucleic acids of DOF transcription factor polypeptide.

Should be understood that suitability of the present invention is not subject to the DOF transcription factor nucleic acid of being represented by SEQ ID NO:226, suitability of the present invention is not subject to the DOF transcription factor expression of nucleic acids by the prolamine promoters driven yet.

The example of seed specific promoters is shown in the table 7 of this specification sheets " definition " part, and described promotor or derivatives thereof can be used for implementing method of the present invention.

Randomly, one or more terminator sequences can also be used for importing the construct of plant." definition " part at this specification sheets has defined term " terminator ".

Gene construct of the present invention also can comprise in order to keep and/or duplicate necessary replication orgin sequence in specific cell type.An example is that gene construct need be as additive type genetic elements (for example plasmid or clay molecule) when remaining in the bacterial cell.Preferred replication orgin includes but not limited to f1-ori or colE1.

Gene construct can randomly comprise the selectable marker gene of this specification sheets " definition " part definition.

The present invention also comprises can be by the plant of method acquisition of the present invention.Therefore the invention provides can be by plant, its plant part or the vegetable cell of method acquisition of the present invention, and described plant or its part or cell comprise the nucleic acid transgenosis (or its variant) as defined above of encoding D OF transcription factor polypeptide.

The present invention also provides and has been used to produce the method for comparing the transgenic plant of the output with increase with suitable control plant, and described method is included in nucleic acid or its variant that imports in the plant and express encoding D OF transcription factor polypeptide.

(i) nucleic acid or its variant of importing and expression DOF transcription factor polypeptide in plant, plant part or vegetable cell; With

Term " conversion " is the conversion of " definition " part definition in this specification sheets.

The present invention also comprises the isolating nucleic acid that comprises encoding D OF transcription factor polypeptide or the host cell of its variant.Preferred host cell of the present invention is a vegetable cell.

The present invention also extends to the part gathered in the crops of plant such as but not limited to seed, leaf, fruit, flower, stem, rhizome, stem tuber and bulb.The invention still further relates to and derive from, preferably be directed to the product of the part gathered in the crops of such plant, for example dried granulated feed or dry powder, oil, fat and lipid acid, starch or protein.

The present invention comprises that also the nucleic acid of encoding D OF transcription factor polypeptide or its variant and DOF transcription factor polypeptide increase the above purposes of the plant biomass of definition in the method for the invention.

The nucleic acid of encoding D OF transcription factor polypeptide or its variant, or DOF transcription factor polypeptide can be used for the procedure of breeding, in the described procedure of breeding, identify can with the dna marker of DOF transcription factor gene or its variant genetic linkage.This nucleic acid/gene or its variant, or DOF transcription factor polypeptide can be used for defining molecule marker.This DNA or protein labeling can be used for the procedure of breeding to select to have the plant of the output that increases as defined above in the method for the invention then.

The allele variant of DOF transcription factor nucleic acid/gene also can be used for the marker-assisted breeding program.This type of procedure of breeding needs sometimes by plant is carried out mutagenic treatment, and for example using, EMS mutagenesis imports allelic variation; Selectively, this program can start from the set of the allele variant in non-so-called " natural " source that produces intentionally.Carry out the evaluation of allele variant by for example PCR then.Select step afterwards, can cause good allele variant output, goal gene that increases with selection.Usually the growth performance of plant that comprises the different allele variants of aim sequence by monitoring is selected.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises and will wherein identify plant and other plant hybridization of good allele variant.This can be used for for example producing the combination of significant phenotypic characteristic.

The nucleic acid of encoding D OF transcription factor polypeptide or its variant also can be used as the mark that gene (described nucleic acid or its variant are the parts of described gene) is carried out the heredity and the probe of physical mapping and conduct and the proterties of these gene linkages.This information can be used for plant breeding has the phenotype of expectation with generation strain.It is the nucleotide sequence of at least 15 Nucleotide that this purposes of DOF transcription factor nucleic acid or its variant only needs length.DOF transcription factor nucleic acid or its variant can be used as restriction fragment length polymorphism (RFLP) mark.Available DOF transcription factor nucleic acid or its variant are surveyed the Southern trace (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of the plant genome DNA of restrictive diges-tion.The program that uses a computer then for example MapMaker people (1987) Genomics 1:174-181 such as () Lander is carried out genetic analysis to make up genetic map with the banding pattern of gained.In addition, can use nuclei acid probe to comprise the Southern trace of the genomic dna of one group of individuality that restriction endonuclease handles, the parent and the offspring of the genetic cross of wherein should the individual representative of group determining.The separation of record dna polymorphism is used it for and is calculated position in the genetic map that uses this colony to obtain before of DOF transcription factor nucleic acid or its variant people (1980) Am.J.Hum.Genet.32:314-331 such as () Botstein.

The generation and the application of the probe in the plant gene source that is used for genetic mapping have been described in Bernatzky and Tanksley (1986) (Plant Mol.Biol.Report 4:37-41).Many publications have been described the genetic mapping that uses aforesaid method or its modification that specific cDNA clone is carried out.For example, the individuality of F2 hybridization colony, backcross population, panmictic population, near isogenic line and other groups can be used for mapping.These class methods are known to those skilled in the art.

Can use nucleic acid to carry out the multiple method that is used for heredity and physical mapping based on nucleic acid amplification.Example comprises the segmental polymorphism (CAPS of allele specific amplification (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplification; People such as Sheffield (1993) Genomics16:325-332), allele-specific connects people (1988) Science241:1077-1080 such as () Landegren, Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radioactivity hybridization mapping people (1997) Nat.Genet.7:22-28 such as () Walter and Happy map (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, it is right that the sequence of nucleic acid is used to design and produce the primer that is used for amplified reaction or primer extension reaction.This type of primer design is known to those skilled in the art.In the method for the genetic mapping that uses PCR-based, may must in corresponding to the zone of this nucleotide sequence, identify the dna sequence dna difference of mapping between the parent of hybridizing.Yet this is optional usually for drawing method.

Method of the present invention causes producing above described plant with output of increase.Also can be somebody's turn to do output and other the economic favourable proterties that increases, for example the proterties of other increase output, combined to the proterties of other abiotic and biological resistances of coercing, modification different structure feature and/or biological chemistry and/or physiological characteristic.

The detailed description of CKI

Preferentially " minimizing " endogenous CKI expression of gene in the albumen tissue of herein mentioning, be used in reference to the endogenous CKI gene expression dose of in the endosperm tissue of wild-type plant, finding and compare, reduce or eliminate basically the expression of endogenous CKI gene (in endosperm tissue).This minimizing of endogenous CKI genetic expression or basically eliminate can cause CKI protein level and/or active minimizing or elimination basically in the albumen tissue.

" endogenous " CKI gene of herein mentioning not only is meant the CKI gene of finding with its natural form (that is, without any artificial intervention) in plant, but also refers to import subsequently the isolating CKI gene of plant.For example, comprise minimizing or the basically eliminate (in endosperm tissue) that the genetically modified transgenic plant of CKI may run into the genetically modified minimizing of CKI or basically eliminate and/or endogenous CKI gene.

Can use one or more methods in several gene silencing methods of knowing, realize this minimizing (or basically eliminate) of endogenous CKI genetic expression." gene silencing " or expression " downward modulation " as used herein, are meant the minimizing or the basically eliminate of CKI genetic expression and/or CKI polypeptide level and/or CKI polypeptide active.

Be used to reduce or such method of the endogenous CKI genetic expression of basically eliminate is the downward modulation (RNA silence) of the genetic expression of RNA mediation.In this case by in plant, triggering reticent with the double stranded rna molecule (dsRNA) of target CKI dna homolog basically.This dsRNA can by plant further be processed into be called short interfering rna (siRAN) about 21 to about 26 Nucleotide.SiRNA is integrated into RNA inductive silencing complex (RISC), the mRNA of this complex body cutting CKI target gene, thus reduce or eliminate the number that remains to be translated into the proteic CKI mRNA of CKI basically.

An example of RNA silencing methods relates to sense orientation encoding sequence or its part importing plant." sense orientation " is meant and its mRNA transcript homologous DNA.Import the DNA of plant thereby will be at least one additional copy (completely or partially) that has been present in the CKI gene in the host plant.This extra gene or its part will cause the silence of endogenous CKI gene, produce the phenomenon that is called common inhibition.If several additional copies are imported plant, the minimizing of CKI genetic expression will be more obvious, and this is because have positive correlation between the triggering of high transcriptional level and inhibition altogether.

Another example of RNA silencing methods relates to use antisense CKI nucleotide sequence." antisense " and nucleic acid comprises " justice is arranged " nucleic acid complementation with proteins encoded, for example with the coding strand of double-stranded cDNA molecule complementary or with the complementation of mRNA sequence, nucleotide sequence.Therefore, antisense nucleic acid can form hydrogen bond with phosphorothioate odn is arranged.Antisense nucleic acid can with whole C KI coding strand or only with its part complementation.Antisense nucleic acid molecule can be to " coding region " or " non-coding region " antisense of coding strand of the nucleotide sequence of coding CKI.Term " coding region " is meant the zone of the nucleotide sequence that comprises the codon of translating into amino-acid residue.Term " non-coding region " be meant be positioned at not translating into of coding region flank amino acid whose 5 ' and 3 ' sequence (that is, be also referred to as 5 ' and 3 ' non-translational region).

Can be according to Watson and Crick base pairing rules design antisense nucleic acid.Antisense nucleic acid molecule can with the whole coding region complementation of CKI mRNA, but it is preferably only to the oligonucleotide of a part of antisense of the coding of CKI mRNA or non-coding region.For example, antisense oligonucleotide can with the regional complementarity around the translation initiation position of CKImRNA.The length of suitable antisense oligonucleotide is known in this area and can starts from the length of about 20 Nucleotide or shorter.Can use chemosynthesis and use the method known in the art to carry out the enzyme ligation, make up antisense nucleic acid of the present invention.For example, the chemosynthesis antisense nucleic acid (for example can to use the Nucleotide of natural generation or various modified Nucleotide (described modified Nucleotide can or increase antisense and the physical stability of the duplex that forms between the phosphorothioate odn is arranged through design in order to the biological stability that increases molecule), antisense oligonucleotide), for example, the Nucleotide that can use phosphorothioate derivative and acridine to replace.The example that can be used for producing the modified Nucleotide of antisense nucleic acid is known in this area.

Other known nucleotide modifications comprise methylate, cyclisation and ' adding cap ' and with analogue for example inosine to the displacement of the Nucleotide of one or more natural generations.Other modifications of Nucleotide are known to those skilled in the art.

Selectively, can use with antisense orientation (that is, be the antisense orientation for the purpose target nucleic acid, further describe in the trifle below) subclone the expression of nucleic acids carrier, produce antisense nucleic acid by biological method from the RNA of the transcribed nucleic acid that inserts.Preferably, the transgenosis by stable integration (described transgenosis comprise for preferentially effective promotor, antisense oligonucleotide and terminator for the expression in the endosperm tissue of plant) produces antisense nucleic acid in plant.

Be used for reducing or a preferred method eliminating endogenous CKI genetic expression basically is to use expression vector by the RNA silence, the form with the inverted repeats (partially or completely) separated by transcribed spacer (noncoding DNA) has been cloned CKI gene or its fragment in this carrier.After inverted repeats is transcribed, form the chimeric CKI RNA that (partially or completely) has self complementary structure.This double-stranded RNA structure is called hairpin RNA (hpRNA).HpRNA is processed into the siRNA that is integrated into RISC by plant.RISC further cuts the mRNA of CKI target gene, thereby reduces or eliminate basically the number of the proteic CKI mRNA of one-tenth CKI to be translated.Referring to for example, people such as Grierson (1998) WO 98/53083; People such as Waterhouse (1999) WO 99/53050).

Be used in the method for the invention reticent nucleic acid molecule (no matter be import plant or original position produces) can or combine with coding proteic cell mRNA of CKI and/or genomic dna hybridization, thereby for example suppress this protein expression by suppressing to transcribe and/or translate.Nucleotide that can be by routine is complementary to be formed stable duplex and realize described hybridization, or for example, under the situation in conjunction with the antisense nucleic acid of DNA duplex, interacts by the specificity in double-helical major groove and to hybridize.Can import antisense nucleic acid molecule by conversion or direct injection at the particular organization position.Selectively, can modify antisense nucleic acid molecule, carry out systemic administration then with the selected cell of target.For example, for systemic administration, antisense molecule can be modified so that they can specificity be combined in the acceptor or the antigen of expressing on the selected cell surface, for example by antisense nucleic acid molecule is realized described modification with can being connected in conjunction with cell surface receptor or antigenic peptide or antibody.Also can use carrier described herein that antisense nucleic acid molecule is delivered to cell.

According to more on the one hand, antisense nucleic acid is a α-different nucleic acid molecule.α-different nucleic acid molecule and complementary RNA form special double-stranded crossbred, and be different with common β unit in described crossbred, and the trend of chain is parallel each other (people (1987) Nucleic Acids.Res.15:6625-6641 such as Gaultier).Antisense nucleic acid molecule also can comprise 2 '-neighbour-methyl ribonucleotides (people (1987) Nucleic Acids Res.15:6131-6148 such as Inoue) or mosaic type RNA-DNA analogue (people (1987) FEBS Lett.215:327-330 such as Inoue).

In another embodiment, antisense nucleic acid of the present invention is a ribozyme.Ribozyme is the catalysis RNA molecule with the ribonuclease activity that can cut the single-chain nucleic acid (for example mRNA) that has complementary district with it.Therefore, ribozyme (for example, hammerhead ribozyme (being described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used for catalyze cleavage CKI mRNA transcript, thereby suppresses the translation of CKImRNA.The nucleic acid that can design coding CKI based on the nucleotide sequence of CKI cDNA has specific ribozyme.For example, can make up the derivative of thermophilas (Tetrahymena) L-19IVS RNA, wherein nucleotide sequence complementation to be cut among the mRNA of the nucleotide sequence of avtive spot and coding CKI.Referring to, people's United States Patent (USP) 4,987,071 such as Cech for example; With people's United States Patent (USP)s 5,116,742 such as Cech.Selectively, can use CKI mRNA from the RNA library of molecules, to select to have the catalysis RNA of specific rna enzymic activity.Referring to, for example, Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.The purposes that ribozyme is used for carrying out plant gene silencing is known (for example, people (1994) WO 94/00012 such as Atkins in this area; People such as Lenne (1995) WO 95/03404; People such as Lutziger (2000) WO 00/00619; People (1997) WO 97/38116 such as people such as Prinsen (1997) WO 97/13865 and Scott).

Also can be by insertion mutagenesis (for example, T-DNA inserts or transposon inserts) or by Angell and Baulcombe 1998 (Amplicon VIGS WO 98/36083); The gene silencing strategy that Baulcombe people such as (WO99/15682) describes obtains gene silencing.

If have sudden change having sudden change on the endogenous CKI gene and/or importing subsequently on the isolating CKI gene of plant, but so also producer silence.Can cause the minimizing or the basically eliminate of CKI expression by non-functional CKI.CKI is in conjunction with CDK and cyclin (people such as Verkest, (2005) Plant Cell 17:1723-1736).For example will provide still can be in conjunction with CDK but can not suppress the CKI of active CDK-cyclin mixture in the sudden change of the cyclin binding site in the CKI.

Another gene silencing methods is to use with control region (for example, CKI promotor and/or enhanser) the complementary nucleotide sequence of CKI and practices shooting, to form the triple-helix structure that stops the CKI gene to be transcribed in target cell.Referring to Helene, C. (1991) Anticancer Drug Des.6 (6): 569-84; Helene, people such as C. (1992) Ann.N.Y.Acad.Sci.660:27-36; And Maher, L.J. (1992) Bioassays 14 (12): 807-15.

The example of the different methods that is used for gene silencing (to reduce or the endogenous CKI genetic expression of basically eliminate) has been described above.Method of the present invention depends on the preferentially expression decreased in the endosperm tissue of plant of endogenous CKI gene.Those skilled in the art will be easily for example by using the above-mentioned method that is used for silence of suitable promoter engineering, to obtain preferential gene silencing in endosperm tissue.

Should be understood that, key point of the present invention is the favourable and astonishing result that is reduced or finds during basically eliminate when the expression of endogenous CKI gene in the albumen tissue, and is not limited to be used for this minimizing of endogenous CKI genetic expression or any ad hoc approach of basically eliminate.Other these class methods are known to those skilled in the art.

For optimum operation, be used to reduce or the gene silent technology of the endogenous CKI genetic expression of basically eliminate requires and will be used for transforming monocots from monocotyledonous CKI nucleotide sequence.Preferably, will import identical species from the CKI nucleic acid of any given plant species.For example, will be transformed into rice plant from the CKI nucleic acid (no matter it is total length CKI sequence or fragment) of rice.Will not import identical plant variety by CKI nucleic acid.

" the CKI gene " herein mentioned or " CKI nucleic acid " are used to represent deoxyribonucleotide polymkeric substance or ribonucleoside acid polymer any length, two strands or strand, or its analogue, wherein said analogue has the essential characteristic of natural ribonucleotide, because they can be with mode similar to the polynucleotide of natural generation and nucleic acid hybridization." CKI gene " or " CKI nucleic acid " is meant that length is enough to carry out the Nucleotide of successive basically in the CKI encoding gene of gene silencing; This length may be as few as 20 or Nucleotide still less.Encoding, (functional) proteic gene reduces for above-mentioned being used to or the whole bag of tricks of the endogenous CKI expression of gene of basically eliminate is optional.

(it can be made up of 20 or still less Nucleotide can to use the Nucleotide of successive basically of sufficient length in CKI gene/nucleic acid, it can be from any part of CKI gene/nucleic acid, for example 3 ' end of very conservative coding region in the CKI gene family) implement the inventive method.

The CKI gene is known in this area, Monsanto Technology LLC under one's name open International Patent Application WO 2005/007829 and CropDesign N.V under one's name disclosed International Patent Application WO 02/28893 and WO 99/14331 in the Nucleotide of successive basically of any plant CKI gene/nucleic acid of describing all can be used for method of the present invention, during these CKI gene/nucleotide sequences intactly are incorporated herein.

Other CKI gene/nucleotide sequences also can be used for method of the present invention, and can easily be identified by those skilled in the art.Can identify the CKI polypeptide by the one or more existence in several features of knowing (referring to following).After identifying the CKI polypeptide, those skilled in the art can use routine techniques easily to obtain the respective coding nucleotide sequence, and use the continuous nucleotide of the sufficient length of described sequence to carry out any one or a plurality of said gene silencing methods (to reduce or the endogenous CKI expression of gene of basically eliminate) in endosperm.

The feature of a uniqueness of CKI polypeptide is to comprise the about 40 amino acid whose C end regions to about 55 high conservatives.As guiding, comprise C end regions at least 50% with the CKI that represents by SEQ ID NO:262 by the preferred sequence that increases progressively, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the polypeptide of 99% identity can be considered the homologue of CKI.Those skilled in the art can easily derive the corresponding nucleic of the described homologue of coding, and use the continuous nucleotide of the sufficient length of described nucleic acid to carry out any one or more said gene silencing methods (to reduce or the endogenous CKI expression of gene of basically eliminate).

Those skilled in the art will very clear proteinic " C end " implication; For the application's purpose, the C end regions of CKI can be considered as back 1/2nd of total length CKI polypeptide (holding end) to C from N.

Can use the routine techniques known in this area for example by sequence alignment, easily identify homologue as defined above, promptly comprise polypeptide with C end regions at least 50% identity of the CKI that represents by SEQ ID NO:262.The sequence alignment method that is used to carry out the sequence comparison is known in this area, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses the comparison of algorithm ((1970) J Mol Biol 48:443-453) to find the making number maximization of coupling and to make minimized two complete sequences of number in room of Needleman and Wunsch.BLAST algorithm people (1990) J Mol Biol 215:403-10 such as () Altschul calculates per-cent sequence identity and carries out the statistical analysis of two similaritys between the sequence.Being used to carry out the software that BLAST analyzes can obtain by American National biotechnology information center publicly.Can use for example can be at the ClustalW multisequencing alignment algorithm (version 1.83) of http://clustalw.genome.jp/sit-bin/nph-ClustalW acquisition, the methods of marking that uses default paired comparison parameter and represent with per-cent is easily identified homologous sequence.Can carry out a small amount of human-edited to optimize the comparison between the conservative primitive (referring to following), this it will be apparent to those skilled in the art that.

Also can come plant identification CKI polypeptide by the existence of some conservative primitive (referring to following table 12).Can use to be used for sequence comparison method relatively as described above, identify the existence of these conservative primitives.In some cases, can adjust default parameter to change the severity of search.For example when using BLAST, can increase the statistical significance threshold value (being called " expectation " value) that is used to report at the coupling of database sequence and show the coupling that severity is lower.By this method, can identify short coupling almost completely.After identifying the CKI polypeptide according to the existence of these primitives, those skilled in the art can easily derive the corresponding nucleic that coding comprises the polypeptide of dependency basis unit, and use the continuous nucleotide of the sufficient length of described nucleic acid to carry out above-mentioned any or several genes silencing methods (to reduce or the endogenous CKI expression of gene of basically eliminate).

Usually, exist (for example primitive 2 is especially very conservative) of at least one should be enough to any search sequence is accredited as CKI in the primitive 1 to 5, yet in order to increase determinacy, primitive 1,2 and 3 existence are preferred at least.The consensus sequence that provides is based on the sequence that shows in the following table 12.Those skilled in the art will be very clear, if use other or different sequences to compare, consensus sequence may slightly change.

Primitive 1:FXXKYNFD (SEQ ID NO:261), wherein X is any amino acid

Primitive 2:[P/L] LXGRYEW (SEQ ID NO:262), wherein X is any amino acid, position shown in [P/L] expression proline(Pro) or leucine appear at.

Primitive 3:EXE[D/E] FFXXXE (SEQ ID NO:263), wherein X is position shown in any amino acid and [D/E] expression aspartic acid or L-glutamic acid appear at.

Primitive 4:YXQLRSRR (SEQ ID NO:264), wherein X is any amino acid,

Primitive 5:MGKY[M/I] [K/R] KX[K/R] (SEQ ID NO:265), wherein X is any amino acid, position shown in [M/I] expression methionine(Met) or Isoleucine appear at, position shown in [K/R] expression Methionin or arginine appear at.

Primitive 6:SXGVRTRA (SEQ ID NO:266), wherein X is any amino acid.

Usually at the proteic carboxyl terminal area discover of plant CKI primitive 1,2 and 3.This zone it is believed that the interaction that participates in CKI and CDK and cyclin (people (1996) Mol.Cell Biol 16 such as Chen, 4673-4682, people such as Matsuoka (1995) Genes Dev.9,650-662, with Nakayama and Nakayama (1998) Bioessays 20,1020-1029).Usually at the proteic aminoterminal area discover of the CKI of plant primitive 4,5 and 6.

From the CKI of monocotyledonous CKI albumen, particularly rice, it is characterized in that the extensively α spiral fragment of existence, particularly between primitive 5 and 6 and between primitive 6 and 4.

Conservative primitive in the table 12. plant CKI albumen.CKI1 to CKI7 represents Arabidopis thaliana CKI.Os: rice, Zm: corn, Sb: Chinese sorghum.

Except above-mentioned feature, CKI albumen also can comprise in following any one or a plurality of: Cy frame (Cy-box), nuclear localization sequence and PEST sequence.

Term " Cy frame " is meant that the length with consensus sequence RXHuF is the aminoacid sequence of about 5 amino-acid residues, and wherein X is any amino acid, and Hu is the uncharged amino acid of hydrophobicity, for example M, I, L or V.The Cy frame participates in the interaction of CKI and cyclin usually.

" nuclear localization sequence " is meant that length is the aminoacid sequence of about 4 to 20 amino-acid residues, and this sequence is used to instruct protein to arrive nucleus.Usually, nuclear localization sequence is rich in basic aminoacids, for example arginine (R) and Methionin (K).For example among Gorlich D. (1998) the EMBO 5.17:2721-7 nuclear localization sequence is being described.Os CKI4 albumen comprises a plurality of nuclear localization sequences.

" PEST sequence " is meant and is rich in amino-acid residue proline(Pro) (P), L-glutamic acid (E), Serine (S) and Threonine (T) and is present in aminoacid sequence in the protein with high protein hydrolysis turnover rate.At people (1986) Science 234 such as for example Rogers, the PEST sequence is disclosed among the 364-368.

Can use for example SMART (people (1998) Proc.Natl.Acad.Sci.USA 95 such as Schultz, 5857-5864 of special database; People such as Letunic (2002) Nucleic Acids Res 30,242-244; Http:// smart.embl-heidelberg.de/), InterPro (people such as Mulder, (2003) Nucl.Acids.Res.31,315-318; Http:// www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecularsequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; The 2nd international conference record Altman R. of molecular biology intelligence system, Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAIPress, Menlo Park; People such as Hulo, Nucl.Acids.Res.32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (people such as Bateman, Nucleic AcidsResearch30 (1): 276-280 (2002), Http:// www.sanger.ac.uk/Software/Pfam/), identify the various structural domains in the CKI albumen.

In addition, also can by suppress cell cycle protein dependent kinase (CDK) for example the active ability of plant CDK identify CKI albumen.CDK regulates for example one group of serine/threonine kinase of cell cycle progression in the plant of eukaryote.CDK is common and cyclin is compound, thereby forms enzyme complex, and wherein CDK is a catalytic subunit, and cyclin is adjusting subunit (Wang, H. (1997) the The Plant Journal 15 (4): 501-510) of enzyme complex.

Therefore after using one or several above-mentioned characterized CKI polypeptide, those skilled in the art can easily derive the corresponding nucleic of this polypeptide of coding, and use the Nucleotide of successive basically of the sufficient length of described nucleic acid to carry out any or multiple said gene silencing methods (to reduce or the endogenous CKI expression of gene of basically eliminate).

For method of the present invention, the preferred Nucleotide of successive basically that uses the sufficient length of SEQ ID NO:267 (OsCKI4), or the sufficiently long continuous nucleotide basically of the nucleotide sequence of the lineal homologue of use coding OsCKI4 (SEQ ID NO:267) or collateral line homologue.Below table 13 this type of the lineal homologue of OsCKI4 and the example of collateral line homologue are provided.

Lineal homologue and collateral line homologue are the homologues that comprises the evolution notion of the ancestral relationship that is used to describe gene.The collateral line homologue is the interior gene that doubles to originate from by ancestral gene of same species, and lineal homologue is to form the gene that originates from from the different biological species that pass through.

Can be by carrying out so-called mutual blast research, easily find the lineal homologue in the monocotyledons species for example.This can by a blast (relate at any sequence library, for example can The public obtainable NCBI that http://www.ncbi.nlm.nih.gov. findsDatabase carries out blast to search sequence (for example, SEQ ID NO:267 or SEQ ID NO:268) and analyzes) carry out.When starting from nucleotide sequence, can use BLASTN or TBLASTX (use standard default), when starting from protein sequence, can use BLASTP or TBLASTN (use standard default).Can randomly filter BLAST result.Then the full length sequence among filtering result or the unfiltered result is carried out reverse BLAST to the sequence of the biology of originating from search sequence and analyze (the 2nd BLAST) (when search sequence is SEQ ID NO:267 or SEQ ID NO:268, therefore the 2nd blast will carry out at the rice sequence).The result who compares first and second BLAST then.If the high-level hit event from the 2nd blast comes from identical species with search sequence, be accredited as the collateral line homologue so; If high-level hit event and search sequence come from species inequality and are accredited as lineal homologue so.High-level hit event is the hit event with low E value.The E value is low more, score remarkable more (or in other words, the probability that chances on this hit event is low more).The calculating of E value is known in this area.Under the situation of extended familys, can use ClustalW, make up then in abutting connection with tree, manifest the cluster of genes involved and identify lineal homologue and collateral line homologue with help.

Lineal homologue and the collateral line homologue (SEQ ID NO:267 and 268) of table 13:OsCKI4

Title	NCBI	SEQ ID nucleotide sequence	SEQ ID peptide sequence	The source
Title	NCBI	SEQ ID nucleotide sequence	SEQ ID peptide sequence	The source	The Zeama_CKI4 sample	AY986792		269	270	Corn
The Triae_CKI4 sample	The contig of BG908519.1 and CA640135.1	271	272	Common wheat	The Zeama_CKI4 sample	AY986792		269	270	Corn
The Triae_CKI4 sample	The contig of BG908519.1 and CA640135.1	271	272	Common wheat	Orysa_CKI3	AK064723.1	273	274	Rice
The Zeama_CKI3 sample	DVI74570.1	275	276	Corn	Orysa_CKI3	AK064723.1	273	274	Rice
The Zeama_CKI3 sample	DVI74570.1	275	276	Corn	The Sorbi_CKI3 sample	The contig of CN152732.1 and CD224882.1	277	278	Chinese sorghum
The Sacof_CKI4 sample	CO373621.1	279	280	Sugarcane	The Sorbi_CKI3 sample	The contig of CN152732.1 and CD224882.1	277	278	Chinese sorghum

The source of the continuous nucleotide basically of CKI gene/nucleic acid can be any plant origin or artificial source.For optimum operation, be used to reduce or the gene silent technology of the endogenous CKI genetic expression of basically eliminate need use from monocotyledonous CKI sequence to be transformed into monocotyledons.Preferably, will be transformed into plant gramineous from CKI sequence gramineous.More preferably, will be transformed into rice plant from the CKI nucleic acid (no matter it is total length CKI sequence or fragment) of rice.Will not import identical plant variety by CKI nucleic acid.More preferably, from the CKI nucleic acid of rice be the continuous nucleotide basically of sufficient length of the nucleotide sequence of the lineal homologue of the continuous nucleotide basically of sufficient length of SEQ ID NO:267 (OsCKI4) or coding OsCKI4 (SEQ ID NO:267) or collateral line homologue.As mentioned above, those skilled in the art will be very clear, in order to carry out above-mentioned any gene silencing methods, what will form the Nucleotide of successive basically of sufficient length, and this sequence can be little of 20 or still less the Nucleotide of successive basically in some cases.

Therefore, gene construct is provided, described gene construct comprises one or more control sequences and transcription termination sequence randomly, and described control sequence can preferentially drive in the albumen tissue has the expression of justice and/or antisense CKI nucleotide sequence with reticent endogenous CKI gene in the endosperm tissue of plant.

A preferred construct that is used for gene silencing comprises CKI gene or its segmental inverted repeats, preferably can form the inverted repeats of hairpin structure, and this inverted repeats is under the control of endosperm specificity promoter.

Aim sequence effectively is connected with one or more control sequences (at least with promotor) that can preferentially increase expression in the endosperm tissue of plant.Term " controlling element ", " control sequence " and " promotor " all are used interchangeably herein, and define in " definition " part.

Endosperm specificity promoter is meant preferentially any promotor of driving purposes genetic expression in endosperm tissue." preferentially " herein mentioned drives expression in endosperm tissue, be used for representing basically in the expression of endosperm tissue driving with its any sequence that effectively is connected, and except any residual expression that causes owing to the seepage promoter expression, the elsewhere of getting rid of plant drives expression.For example, the prolamine promotor shows strongly expressed in endosperm, and at meristematic tissue, more preferably has leakage expression among stem meristematic tissue and/or the merismatic discrimination centre.

Preferably, endosperm specificity promoter is the promotor from the prolamine gene isolation, the paddy prolamine RP6 that represents by SEQ ID NO:281 (people such as Wen, (1993) Plant Physiol101 (3): 1115-6) promotor or have promotor to this similar intensity of paddy prolamine promotor and/or similar expression pattern for example.Can for example pass through promotor and reporter gene coupling, the function of examining report gene is analyzed similar intensity and/or similar expression pattern in the tissue of plant then.Well-known reporter gene is the colorimetric GUS dyeing that β-glucuronidase and being used for manifests the β-glucuronidase activity of plant tissue.The example that also can be used for implementing other endosperm specificity promoters of method of the present invention is shown in table 6 of " definition " part.

The construct that randomly, also one or more terminator sequences can be used for plant to be imported.Term " terminator " is as the definition of " definition " in this specification sheets part.

Gene construct of the present invention also can comprise in order to keep and/or duplicate necessary replication orgin sequence in specific cell type.An example is need be with in additive type genetic elements (for example plasmid or clay molecule) the maintenance gene construct in bacterial cell.Preferred replication orgin includes but not limited to f1-ori and colE1.

Gene construct can randomly comprise the selectable marker gene of " definition " part definition in this specification sheets.

The present invention also comprises can be by the plant of method acquisition of the present invention, comprise plant part, described plant is compared the seed production with increase with suitable control plant and have endogenous CKI expression of gene minimizing or that eliminate basically in the albumen tissue.

The present invention also provides and has been used to produce the transgenic plant of comparing the seed production with increase with suitable control plant, and described transgenic plant have the endogenous CKI expression of gene that reduces or eliminate basically in the albumen tissue.

(i) in plant, plant part or vegetable cell, import and express the gene construct that comprises one or more control sequences, described control sequence can be preferentially drives in the albumen tissue has justice and/or antisense CKI nucleotide sequence to express, thus in the endosperm tissue of plant reticent endogenous CKI gene; With

(ii) under the condition that promotes plant-growth and development of plants, cultivate this plant, plant part or vegetable cell.

Preferably, the construct that imports plant comprises CKI gene or its segmental inverted repeats (partially or completely), preferably can form the inverted repeats of hairpin structure.

According to preferred feature of the present invention, construct is imported plant by transforming.

Term " conversion " is the conversion that defines in " definitional part " of this specification sheets.

The present invention clearly extends to any vegetable cell or the plant that produces by any method in the method described herein and extends to its all plant parts and its propagulum.The present invention extends further to and comprises that the former generation conversion that produces by any method in the aforesaid method or the offspring of cells transfected, tissue, organ or complete plant, unique requirement are that this offspring shows genotype and/or identical genotype and/or the phenotypic characteristic of phenotypic characteristic with parent's generation in the method for the invention.

The part gathered in the crops that the present invention also extends to plant is seed and deriving from for example, preferably is directed to the product of the part gathered in the crops of such plant, for example dried granulated feed or dry powder, oil, fat and lipid acid, starch or protein.

The present invention comprise also that CKI nucleic acid is used for reducing or the endogenous CKI gene of basically eliminate in the expression of albumen tissue to increase the above purposes of the plant seed output of definition.

Accompanying drawing is described

With reference to following accompanying drawing the present invention is described, wherein:

Fig. 1 provides the general introduction that is present in the conservative primitive on the SEQ ID NO:2.Be rich in leucic structural domain and indicate with underscore, conservative primitive 1,2 and 3 shows with runic, represents protein kinase C phosphorylation site of inferring and the N glycosylation site of inferring with the sequence that italic is represented.

Fig. 2 shows that the proteic multiple ratio of multiple SYR is right.Identical amino-acid residue represented in asterisk, and colon is represented the displacement of high conservative, the displacement that the some expression is not too cautious.According to information, can easily in other SYR albumen, identify various structural domains and conservative primitive among the SEQ ID NO:2 from Fig. 1.

Fig. 3 shows the binary vector that is used for transforming and expressing rice rice SYR nucleic acid.In pGOS2::SYR, the SYR encoding sequence is under the control of rice GOS2 promotor.

Fig. 4 shows the binary vector that is used for transforming and expressing rice rice SYR nucleic acid.In pHMGP::SYR, the SYR encoding sequence is under the control of rice HMGP promotor (the SEQ ID NO:18 of WO 2004/070039, this SEQ ID NO:18 among the WO 2004/070039 intactly incorporates into herein).

Fig. 5 has described the example of the sequence that can be used for implementing method of the present invention.SEQ ID NO:1 and SEQ ID NO:2 represent to be used for Nucleotide and the protein sequence of the SYR of embodiment.Initial sum terminator codon among the SEQ IDNO:1 shows with runic.SEQ ID NO:3 is the primer sequence that is used to separate this SYR nucleic acid with SEQ ID NO:4.SEQ ID NO:5 is the sequence of GOS2 promotor, and SEQ ID NO:33 is the sequence of the PRO0170 promotor used among the embodiment, and SEQ IDNO:6 to SEQ ID NO:11 represents the consensus sequence of conservative part in the SYR albumen.SEQ IDNO:12 to 25,27 to 32 and 36 to 42 is the SYR gene that provides in SEQ ID NO:1 and SEQ ID NO:2 and the Nucleotide (total length or part) and the protein sequence of proteinic homologue.SEQ ID NO:26 represents ARGOS protein sequence (GenBank accession number AY305869).

Fig. 6 has provided the general introduction of FG-GAP protein structure domain.The albumen of SEQ ID NO:46 comprises secretion signal (the N end parts of showing with collimation mark), start from P73 and terminate in the FG-GAP structural domain and the membrane spaning domain (showing with runic and collimation mark) that indicate with runic and underscore of L98.Conservative primitive DXDXDGXX (D/E) indicates with frame and underscore, and wherein primitive DGXX (D/E) indicates with italic.Conservative FDGYLYLID structural domain indicates with underscore.

Fig. 7 shows that the multiple ratio of total length FG-GAP albumen (SEQ ID NO:46, SEQ ID NO:55, SEQ IDNO:57 and SEQ ID NO:59) is right, and identical amino acid represented in asterisk, and colon is represented the displacement of high conservative, the displacement that the some expression is not too cautious.The partial sequence of listing among the embodiment 12 table G can be used for this multiple ratio to identify other primitive.

Fig. 8 shows the binary vector that is used for transforming and being expressed in rice the nucleic acid of the coding Arabidopis thaliana FG-GAP under the control of rice GOS2 promotor.

Fig. 9 describes the example of the sequence can be used for implementing method of the present invention.SEQ ID NO:45 and SEQ ID NO:46 represent to be used for Nucleotide and the protein sequence of the FG-GAP of embodiment; Initial sum terminator codon among the SEQ IDNO:45 indicates with runic.SEQ ID NO:47 is the primer sequence that is used to separate FG-GAP nucleic acid with SEQ ID NO:48.SEQ ID NO:49 is the sequence that is used for promotor-assortment of genes of embodiment, and SEQ ID NO:50 to SEQ ID NO:53 represents the consensus sequence of the conservative part in the FG-GAP protein.SEQ ID NO:54 to 71 is the FG-GAP gene that provides in SEQ ID NO:45 and SEQ ID NO:46 and the Nucleotide (total length or part) and the protein sequence of proteinic homologue.SEQ ID NO:72 is the proteic genome sequence of coding alfalfa (Medicagosativa) FG-GAP, and this albumen comprises the peptide sequence by SEQ ID NO:72 to 76 expression.

Figure 10 is presented at the key character of finding in CYP90B polypeptide or its homologue: N end hydrophobic domains, translocation domain (having K/R-K/R-X3-9-P-G-G), A to D structural domain.In the A structural domain, identified consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser.The consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser of CYP90B polypeptide comprises this consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser.

Figure 11 shows the biosynthetic pathway of ramose rape sterol.In Arabidopsis, the CYP90B1/DWF4 polypeptide comprises steroid 22-α hydroxylase enzymatic activity.

Figure 12 shows the hydrophobicity ProtScale output spectra of CYP90B polypeptide of the present invention.Preceding 34 N terminal amino acids (showing with collimation mark) are hydrophobic domains, because these amino acid are positioned on 0 boundary line.This zone is corresponding to N end anchor structure territory.

Figure 13 show use based on improved ClustalW algorithm (InforMax, Bethesda, MD, Http:// www.informaxinc.com) VNTI AlignX multiple ratio to program (use default setting: the open point penalty in room be 10 and to extend point penalty be 0.05 in the room), the multiple ratio that several plant CYP90B polypeptide is carried out is right.Having indicated N holds hydrophobic domains, translocation domain (to have K/R-K/R-X _3-9-P-G-G) and A to D structural domain.In the A structural domain, show consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser with collimation mark.The accession number of CYP90B polypeptide is found in table 9a and 9b.Arabidopis thaliana Arath_CYP90A1_CPD (At5g05690), Arath_CYP90C1_ROT3 (At4g36380) and Arath_CYP90D1 (At3g13730) are shown as non-CYP90B polypeptide.

Figure 14 shows the plant conversion carrier that is used for being expressed in rice the rice CYP90B nucleic acid under the plant promoter control, and described plant promoter can be non-constitutive promoter (for example endosperm or embryo/aleuron are specific) or constitutive promoter (for example GOS2 and HMGB1).

Figure 15 has described the example of the sequence that is used to implement method of the present invention.Several sequences produce from public EST splicing (EST assembly) (referring to table 9a), and it has low-qualityer sequencing.Therefore, can expect and have the minority replacement nucleic acid.When sequence was total length, initial (ATG) and terminator codon defined this nucleotide sequence.

Figure 16 represents the synoptic diagram of total length CDC27 polypeptide (more specifically Arabidopis thaliana CDC27B hobbit polypeptide).34 peptide repeating units (TPR) show with the collimation mark of black.The NH of polypeptide ₂End regions indicates with black bar.

Figure 17 show based on improved ClustalW algorithm (InforMax, Bethesda, MD, Http:// www.informaxinc.com) use VNTI AlignX multiple ratio to program (use default setting: the open point penalty in room be 10 and to extend point penalty be 0.05 in the room) multiple ratio from the CDC27 polypeptide of different sources of carrying out is right.Stride this comparison zone and show 34 peptide repeating units (TPR) with collimation mark.Conservative NH ₂Structural domain PD011373 (as at ProDom, Http:// ribosome.toulouse.inra.fr/ Prodom/current/cgi-bin/ProDomBlast3.plMiddle definition) indicate with double underline.

Figure 18 shows the binary vector pOSH1::CDC27 that is used for being expressed in rice the modified Arabidopis thaliana CDC27 nucleic acid under plant promoter (described promotor the is a stem end meristematic tissue promotor) control.

Figure 19 show by TIGR (Institute for Genomic Research at http://www.tigr.org) produce from the part of different sources and the tabulation of lineal homologue of total length CDC27 and collateral line homologue.TC895803 is found in Http:// www.tigr.org/tigr-scripts/tgi/ Ego/ego report.pl? ego=895803

Figure 20 describes and can be used for the example implementing the sequence of method of the present invention or can be used for separating the sequence of this sequence.Several sequences produce from public EST splicing (referring to table 10), and described sequence has low-qualityer sequencing.Therefore, can expect and have the minority replacement nucleic acid.When sequence encoding total length CDC27 polypeptide, initiator codon (ATG) and terminator codon define this nucleotide sequence.

Figure 21 shows the genealogical tree that comprises the AT hook structure territory and the different peptide sequences of DUF296 structural domain.Based on improved ClustalW algorithm (InforMax, Bethesda, MD, http://www.informaxinc.com) use VNTI AlignX multiple ratio to program (use default setting: the open point penalty in room be 10 and to extend point penalty be 0.05 in the room) produce genealogical tree.

Figure 22 shows and to be used for being expressed in the binary vector pPROLAMIN::AT hook of rice nucleic acid that coding under the control of prolamine promotor comprises the polypeptide of AT hook structure territory and DUF296 structural domain and primitive 2 rice.

Figure 23 show based on improved ClustalW algorithm (InforMax, Bethesda, MD, Http:// www.informaxinc.com) use VNTI AlignX multiple ratio to program (use default setting: the open point penalty in room be 10 and to extend point penalty be 0.05 in the room) multiple ratio of the polypeptide that comprises AT hook structure territory and DUF296 structural domain that carries out is right.In comparison with Runic, italic and underscoreShow AT hook structure territory and DUF296 structural domain and primitive 2.

Figure 24 has described the example of the sequence that is used to implement method of the present invention.

Figure 25 shows the genealogical tree of DOF transcription factor.The frame at close top shows with SEQ IDNO:227 to have homology (with the feature (i) that comprises above definition and (iii), the sequence identity of the DOF structural domain of promptly SEQ ID NO:200 or SEQ ID NO:228 being represented at least 60%; Above Ding Yi primitive I and/or primitive II) the main cluster (major clustering) of sequence.The frame of close bottom shows with SEQ ID NO:199 to have homology (with the feature (i) that comprises above definition and (ii), the sequence identity of the DOF structural domain of promptly SEQ ID NO:200 or SEQ ID NO:228 being represented at least 60%; The main cluster of the sequence sequence identity with the DOF structural domain at least 70% that SEQ ID NO:200 is represented).

Figure 26 shows the binary vector pGOS2::DOF be used for being expressed in rice the Arabidopis thaliana DOF transcription factor under the control of GOS2 promotor.

Figure 27 shows the binary vector pPROLAMIN::DOF that is used for being expressed in rice the Arabidopis thaliana DOF transcription factor under the control of prolamine promotor.

Figure 28 has described the example of the sequence that is used to implement method of the present invention.

Figure 29 is the synoptic diagram of total length plant CKI polypeptide.The typical primitive 1 to 5 (SEQ ID NO:261 to SEQ ID NO:265) that is used to identify CKI shows with collimation mark and correspondingly is numbered (primitive 6 does not show).

Figure 30 show from the multiple ratio of the CKI polypeptide of different sources to use can The ClustalW that http://clustalw.genome.jp obtainsThe adjacency tree that common software (use default setting) produces.The subclass of braces identifying sheet list cotyledon and dicotyledons CKI4.In this subclass, unifacial leaf CKI gathers together, indicates as bracket.Round bracket identifying sheet list cotyledon plant CKI4 branch.

Figure 31 be based on improved ClustalW algorithm (InforMax, Bethesda, MD, Http:// www.informaxinc.com) use VNTI AlignX multiple ratio right to the multiple ratio from the CKI polypeptide of different plant origins that program (use default setting: the open point penalty in room is that 10 to extend point penalty with the room be 0.05) produces.The conservative C end of CKI primitive 1 to 5 (SEQ ID NO:261 to SEQ ID NO:265) terminal and that be used for plant identification CKI shows (primitive 6 does not show) with collimation mark.

Figure 32 is presented at the binary vector that carries out CKI RNA silence in the rice, wherein uses under endosperm specificity promoter is controlled and the hair clip construct under the control of stem specificity promoter.

Figure 33 has described and has been used to the example implementing the sequence of method of the present invention or be used to separate the sequence of described sequence.Several sequences are produced by public EST splicing, and it has low-qualityer sequencing.Therefore, can expect and have the minority replacement nucleic acid.When nucleic acid sequence encoding total length CKI polypeptide, initial (ATG) and terminator codon define this nucleotide sequence.Yet 5 ' and 3 ' UTR also can be used for implementing method of the present invention.

Embodiment

Refer now to and only be used to illustrate purpose the following example and describe the present invention.The following example is not intended to determine fully or otherwise limit scope of the present invention.

The DNA operation

Unless otherwise noted, according to (Sambrook (2001) Molecular Cloning:alaboratory manual, the 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York) or people (1994) such as Ausubel, Current Protocols in Molecular Biology, Current Protocols ( Http:// www.4ulr.com/products/currentprotocols / index.html) the 1st and 2Recombinant DNA technology is carried out in the standard method of describing in the volume.In the Plant Molecular Biology Labfax (1993) that the R.D.D.Croy that is published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications (UK) writes, standard material and the method that is used for the plant molecular operation described.

Statistical analysis

With regard to imbalance design and gauged dual factors ANOVA (variance analysis) as the statistical models of net assessment plant phenotype feature.Parameter with all measurements of all plants of all incidents of this gene transformation is carried out the F check.Carry out the F check with the effect of checking all transformation events of gene pairs and the population effect (being also referred to as " overall genetic effect (global gene effect) " herein) of verifying gene.If the value display data of F check is significant, then draw the conclusion that has " gene " effect, that is, mean to be not only that the existence or the position of gene causes effect.For this F check, the significance threshold setting of real overall genetic effect is on 5% probability level.

Be the genetic effect in the inspection incident, that is, the strain specificity effect is used the data set from transgenic plant and corresponding invalid plant (null plant), carries out the t check in each incident." invalid plant " or " invalid segregant " or " invalid zygote " are the plants of handling in the mode identical with transgenic plant, but transgenosis is separated from this plant.The feminine gender that invalid plant also can be described to isozygoty transforms plant.The significance threshold setting of T check is on 10% probability level.The result of some incidents can be higher or lower than this threshold value.This is based on hypothesis: gene may only have effect on genomic some position, and this position dependence effect is not rare.The genetic effect of the type is also referred to as " the strain effect of gene " herein.By with the t value and t-distribution compares or selectively by F value and F-distribution are compared, obtain the p value.To provide null hypothesis (promptly not having genetically modified effect) be correct probability to the p value then.

Embodiment A: SYR

Embodiment 1: the evaluation of the sequence relevant with SEQ ID NO:2 with SEQ ID NO:1

Use (BLAST) (people (1990) J.Mol.Biol.215:403-410 such as Altschul of for example basic local comparison instrument of database search instrument (Basic Local AlignmentTool); With people (1997) Nucleic Acids Res.25:3389-3402 such as Altschul), in the sequence of the Entrez of American National biotechnology information center (NCBI) Nucleotide database maintenance, identify sequence (full-length cDNA, EST or genome sequence) relevant and/or the protein sequence relevant with SEQ ID NO:2 with SEQ IDNO:1.Usually blast program is by comparing nucleic acid or peptide sequence with sequence library, and by calculating the significance,statistical that mates, is used to seek the zone that has local similar between the sequence.To nucleic acid SEQ ID NO:1 encoded polypeptide utilization TBLASTN algorithm, use default setting, open strainer, to ignore the low complex degree sequence.The output form of analyzing is for comparing in twos, and sorts according to probability score (E value), and wherein score value reflects the occurrent probability of specific comparison (the E value is low more, and the significance of hit event is high more).Except the E value, also keep the score to relatively carrying out identity per-cent.Identity per-cent is meant that two compare the number of the identical Nucleotide (or amino acid) on length-specific between nucleic acid (or polypeptide) sequence.In some cases, can adjust the strict degree of default parameter with the change search.

Except the public obtainable nucleotide sequence that can on NCBI, obtain, also can search for other sequence libraries according to method same as the above-mentioned method.

Table A provides nucleotide sequence of representing with SEQ ID NO:1 and relevant nucleic acid and the protein sequence of representing with SEQ ID NO:2 of protein sequence.

Table A: can be used for the inventive method, with the relevant nucleotide sequence of nucleotide sequence (SEQ ID NO:1), and the corresponding polypeptide of deriving.

Title	The source is biological	Nucleic acid SEQ IDNO:	Polypeptide SEQID NO:	Database login number	State
Title	The source is biological	Nucleic acid SEQ IDNO:	Polypeptide SEQID NO:	Database login number	State	OsSYR	Rice
	1	2	/	Total length or part		OsSYR	Rice
	1	2	/	Total length or part	Rice SYR homologue 1	Rice	12	27	XP_472637	Total length
Rice SYR homologue 2	Rice	13		AP008218	Rice SYR homologue 1	Rice	12	27	XP_472637	Total length	Total length
Rice SYR homologue 2	Rice	13		AP008218	Corn SYR homologue	Zea mays	14	28	AY110705	Part	Total length
Wheat SYR homologue	Common wheat	15		/	Corn SYR homologue	Zea mays	14	28	AY110705	Part	Total length
Wheat SYR homologue	Common wheat	15		/	Barley SYR homologue	Barley	16	36	CB871444	Total length	Total length
Sugarcane SYR homologue 1	Sugarcane	17	37	CA165713	Barley SYR homologue	Barley	16	36	CB871444	Total length	Part
Sugarcane SYR homologue 1	Sugarcane	17	37	CA165713	Sugarcane SYR homologue 2	Sugarcane	18	38	CA242805	Total length	Part
Chinese sorghum SYR homologue	Chinese sorghum	19	39	CX611532	Sugarcane SYR homologue 2	Sugarcane	18	38	CA242805	Total length	Total length

AtSYR homologue 1	Arabidopis thaliana	20	40	NM_115853	Total length
AtSYR homologue 1	Arabidopis thaliana	20	40	NM_115853	Total length	AtSYR homologue
2	Arabidopis thaliana	21	41	NM_180078	Total length	AtSYR homologue
2	Arabidopis thaliana	21	41	NM_180078	Total length	Grape SYR homologue	Grape (Vitis vinifera)	22	29	CF404276	Total length
Oranges and tangerines SYR homologue	Tangerine (Citrus reticulata)	23	30	CF830612	Part	Grape SYR homologue	Grape (Vitis vinifera)	22	29	CF404276	Total length
Oranges and tangerines SYR homologue	Tangerine (Citrus reticulata)	23	30	CF830612	Part	Tomato SYR homologue 1	Tomato	24	32	AI774560	Total length
Tomato SYR homologue 2	Tomato	25	31	BG125370	Total length	Tomato SYR homologue 1	Tomato	24	32	AI774560	Total length

Embodiment 2: the comparison of related polypeptide sequence

From the AlignX of Vector NTI (Invitrogen) based on the clustering algorithm (Clustal algorithm) of the progressive comparison of generally using (people (1997) Nucleic Acids Res25:4876-4882 such as Thompson; People such as Chenna (2003) .Nucleic Acids Res 31:3497-3500).Can use in abutting connection with the clustering algorithm constructing system and set.The default value of the open point penalty in room is 10, and the default value that point penalty is extended in the room is 0.1, and the weight matrix of selection is Blosum 62 (if comparison polypeptide).

Can be used for implementing in the polypeptide of the inventive method in evaluation, the multisequencing comparison result that uses related polypeptide to obtain is shown in Table 2.Can in these different sequences, easily pick out and be rich in leucic tumor-necrosis factor glycoproteins and conservative primitive.

Embodiment 3: calculate the overall per-cent identity (global percentage identity) between the peptide sequence can be used for implementing the inventive method

A method MatGAT in the method that use can obtain in this area (matrix overall comparison instrument (Matrix Global Alignment Tool)) software (BMC Bioinformatics.20034:29.MatGAT:an application that generates similarity/identity matricesusing protein or DNA sequences.Campanella JJ, Bitincka L, Smalley J; Software by Ledion Bitincka trustship), determine to be used to implement overall similarity and identity per-cent between the full-length polypeptide sequence of method of the present invention.MatGAT software need not data are compared in advance, can produce the similarity/identity matrix of DNA or protein sequence.This program is utilized Myers and Miller overall comparison algorithm, and (the open point penalty in room is 12, and to extend point penalty be 2 in the room) carry out a series of comparison in twos, utilize for example Blosum 62 (for polypeptide) calculating similarity and identity, then the result is arranged in distance matrix.Sequence similarity is shown in the diagonal lines Lower Half, and sequence identity is shown in the diagonal lines first half.

More used parameter has:

Matrix: Blosum 62 keeps the score

First room: 12

Extend the room: 2

The overall similarity of peptide sequence length range (the part of polypeptide sequence is foreclosed) and the software analysis of identity the results are shown in table B.The diagonal lines top provides identity per-cent, and the diagonal lines below provides similarity per-cent.

Compare with SEQ ID NO:2, the per-cent identity that can be used for implementing between the peptide sequence of the inventive method can be for being low to moderate 27% amino acid identity.

Embodiment 4: can be used for implementing the topology prediction of the peptide sequence of the inventive method

Utilize the Subcellular Localization of TargetP 1.1 prediction eukaryotic proteins.According to this program, the predictability existence based on following arbitrary N-end presequence positions definite: chloroplast transit peptides (cTP), Mitochondrially targeted peptide (mTP) or Secretory Pathway signal peptide (SP).Final forecasting institute based on score value be not real probability, and add up and needn't be 1.But, according to TargetP, the location that score is the highest is most probable, and the relation between the score value (reliability class) can be used as the index of described forecasting reliability.Reliability class (RC) scope from 1 to 5, the wherein the strongest prediction of 1 expression.TargetP is by the server maintenance of Technical University Of Denmark (Technical University of Denmark).

Also may there be the potential cleavage site in sequence for contain N-end presequence through prediction.

Selected multiple parameter, the prediction and calculation of biological example body type (non-plant or plant), cutoff value setting (the specified cutoff value setting of cutoff value setting of not having, predesignating or user) and cleavage site (be or not).

TargetP 1.1 analytical resultss of peptide sequence shown in the SEQ ID NO:2 are shown among the following table C.What select is " plant " organism type, does not stipulate cutoff value, and requires the prediction length of transit peptides.According to this result, the Subcellular Localization of peptide sequence may be a plastosome shown in the SEQ ID NO:2; Yet should consider 5 reliability class (that is, minimum reliability class).

The TargetP 1.1 of peptide sequence analyzes shown in the table C:SEQ ID NO:2

Length (AA)	105
Length (AA)	105	Chloroplast transit peptides	0.025
The mitochondrial transport peptide	0.552	Chloroplast transit peptides	0.025
The mitochondrial transport peptide	0.552	The Secretory Pathway signal peptide	0.009
Other ubcellular targets	0.416	The Secretory Pathway signal peptide	0.009
Other ubcellular targets	0.416	The location of prediction	Plastosome
Reliability class		The location of prediction	Plastosome
Reliability class		5

Identify two membrane spaning domains by the TMHMM program of trustship on the server at Technical University Of Denmark biological sequence analysis center.Following result shows that it is 0.997 that the N end is positioned at inboard probability.Provided further describing among the table D below about this orientation.

The result of table D:TMHMM2.0

Orientation	Initial-the termination residue
Orientation	Initial-the termination residue	Inboard
	142	Inboard
	142	Transbilayer helix (TMhelix)	4365
The outside	6674	Transbilayer helix (TMhelix)	4365
The outside	6674	Transbilayer helix	7592
Inboard	93105	Transbilayer helix	7592

Can utilize many other algorithms to carry out such analysis, comprise:

● ChloroP 1.1, by Technical University Of Denmark's trust server;

● protein is sought Subcellular Localization forecasting software (Protein Prowler SubcellularLocalisation Predictor), 1.2 version, by (the Institute for Molecular Bioscience of molecular biosciences institute of Brisbane ,Australia University of Queensland, University ofQueensland, Brisbane, trust server Australia);

● PENCE Proteome Analyst PA-GOSUB 2.5, by Canada big Alberta Edmonton Alberta university (University of Alberta, Edmonton, Alberta, trust server Canada);

Embodiment 5: gene clone

(Invitrogen, Paisley UK) pass through pcr amplification rice SYR gene as template to use rice seedling cDNA library.Behind the RNA that reverse transcription is extracted from seedling, cDNA is cloned into pCMVSport 6.0.The average inset size in library is 1.5kb, and clone's original number approximately is 1.59 x10 ⁷Cfu.At 6 x 10 ¹¹After the amplification first time of cfu/ml, initial titer is confirmed as 9.6 x 10 ⁵Cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.Primer prm08170 (the SEQ ID NO:3 that will comprise the AttB site that is used for the Gateway reorganization; 5 '-ggggacaagtttgtacaaaaaagcag gcttaaacaatggaaggtgtaggtgctagg-3 ') and prm08171 (SEQ ID NO:4 justice is arranged, and initiator codon indicates with runic, and the AttB1 site indicates with italic:; Oppositely, complementation; The AttB2 site indicates with italic: 5 '-ggggaccactttgtacaagaaagctgggtcaaaaacaaaaataaattcccc-3 ') be used for pcr amplification.Under standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.Also use the PCR fragment of the correct size of standard method amplification and purifying.Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, and " clone (entry clone) crosses the threshold " pSYR.Plasmid pDONR201 conduct

The part of technology is available from Invitrogen.

Embodiment 6: the structure of carrier

The clone pSYR that will cross the threshold subsequently is used from the LR reaction with the purpose carrier (destination vector) that is used for the rice conversion.This carrier form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and be intended to be used for carry out the Gateway box of recombinating the LR body in the aim sequence that has been cloned in the clone that crosses the threshold.The rice GOS2 promotor (SEQ ID NO:5) that is used for constitutive expression is positioned at the upstream of this Gateway box.Preparation similarly but have high mobility group protein promotor (HMGP, SEQ ID NO:33) but not the vector construction body of GOS promotor.

After the LR reconstitution steps, it is LBA4044 that the expression vector pGOS2::SYR (having the GOS2 promotor) of gained and pHMGP::SYR (having the HMGP promotor) (both is used for composing type SYR and expresses (Fig. 2)) are transformed into Agrobacterium (Agrobacterium) strain, is transformed into rice plant then.

Embodiment 7: the conversion of rice

Use comprises the Agrobacterium-mediated Transformation rice plant of expression vector.Make the ripe dry seeds shelling of rice growing kind Japan fine (ricejaponica cultivar Nipponbare).By in 70% ethanol, hatching 1 minute, then at 0.2%HgCl ₂In hatched 30 minutes, with sterile distilled water washing 6 times, carried out disinfection in each 15 minutes afterwards.Comprising 2 then, sprouting aseptic seed in the substratum of 4-D (callus inducing medium).Incubation cuts the embryo generation callus in scutellum source after 4 weeks in the dark, breeds on identical substratum then.After 2 weeks, breed in other again 2 weeks or breed callus by uploading at identical substratum to be commissioned to train to support.Uploaded for culturing embryo generation callus lines (to strengthen the cell fission activity) at fresh culture in preceding 3 days cultivating altogether.

The Agrobacterium strain that comprises expression vector is that LBA4404 is used for common cultivation.Agrobacterium is seeded in has on the suitable antibiotic AB substratum, cultivated 3 days down at 28 ℃.Collect bacterium then, it is suspended in liquid is total in the culture medium until the density (OD that is approximately 1 ₆₀₀).Then suspension is transferred to culture dish (Petri dish), callus is immersed in the suspension 15 minutes.Then callus is blotted on filter paper, be transferred to altogether culture medium of solidified afterwards, incubation 3 days in the dark under 25 ℃.Then under the situation that selective agent exists under 28 ℃ in the dark, comprising 2,4 weeks of callus that growth is cultivated altogether on the substratum of 4-D.During this period, produced the resistant calli island of quick growth.With this substance transfer to regeneration culture medium with after hatching under the illumination, embryo generation potentiality are released, and grow in ensuing 4 to 5 weeks and sprout.Bud is downcut from callus, in the cultivation that comprises growth hormone, hatched for 2 to 3 weeks then, then with they from described media transfer to soil.In the greenhouse, under high humidity and short day, grow the bud of hardening.

Produce about 35 T0 rice transformant independently for a construct.With former generation transformant be transferred to the greenhouse from tissue culture room.Behind the quantitative PCR analysis of the copy number of confirming the T-DNA inset, only keep and show the results that single copy transgenic plant of selective agent tool resistance are used for the T1 seed.Then after transplanting 3 to 5 months results seed.This method produces single locus transformant (Aldemita and Hodges1996, people such as Chan 1993, people such as Hiei 1994) with more than 50% ratio.

About the conversions of other farm crop referring to embodiment 40.

Embodiment 8: the appraisal procedure of using the SYR plant transformed under rice GOS2 promotor or the control of HMGP promotor

Assessment is provided with

Produced about 15-20 independently T0 rice transformant.In former generation,, transformant transferred to greenhouse growth and results T1 seed by tissue culture room.8 incidents are kept, and wherein T1 separates for the 3:1 that transgenosis existence/shortage takes place.By the expression of monitoring visable indicia, in these incidents, respectively select about 10 T1 seedling that contain transgenosis (heterozygote and homozygote), and about 10 T1 seedling that lack transgenosis (invalid zygote).The T1 plant of selecting is transferred to the greenhouse.Each strain plant is all accepted unique bar code label to make phenotypic data related with corresponding plant clearly.The T1 plant selected is provided with at following environment is grown in down in the soil of flowerpot that diameter is 10cm, described environment setting is: photoperiod=11.5h, intensity of illumination=30,000lux or stronger, daytime temperature=28 ℃ or higher, nocturnal temperature=22 ℃, relative humidity=60-70%.Transgenic plant and corresponding invalid zygote be growth side by side on random site.From sowing time to the ripening stage, plant is repeatedly by the digital image-forming case.On each time point, every strain plant is obtained digital image (2048 x, 1536 pixels, 1,000 6 hundred ten thousand pigments) from least 6 different angles.

The salt stress screening

On the matrix of making by coconut fiber and argex (3 to 1 ratios), cultivate plant from 4 incidents (T2 seed).Sprigging first two weeks to the greenhouse is being used normal nutritive medium.After first two weeks, in nutritive medium, add the salt (NaCl) of 25mM, till the results plant.

The arid screening

In flowerpot soil, cultivate plant under normal operation, up to entering heading stage from 5 incidents (T2 seed).Then it is transferred to " drying " zone, stop to irrigate.In the flowerpot of selecting at random, insert the humidity detection instrument, with monitoring soil water content (SWC).When SWC is lower than certain threshold value, continue moisturizing from the trend plant, up to reaching normal level once more.Then plant is transferred under the normal condition once more again.Remaining cultivation (plant maturation, seed results) is identical with the plant of not cultivating under the abiotic stress condition.With the T2 seed of the plant under normal condition, cultivated results, and need not repeat this screening to carry out taking turns checking from the T2 seed of the plant results of arid screening first.

The parameter of measuring

Plant on the ground area (leaf biomass in other words) is determined by the sum that is different from the pixel of background on the digital image of counting plant part on the ground.This value is got the mean value of same time point from the photo of different angle shots, and is converted to the physical surface value of representing with square millimeter (physical surface value) by calibration.Experiment shows that the ground plant area of measuring by this method is relevant with the biomass that plant shoot divides.Areamax is the ground area when plant reaches its maximum leaf biomass.

Results, packed, with bar code label sophisticated main panicle, under 37 ℃ in baking oven dry 3 days then.To the panicle threshing, collect all seeds then.Use gas blower (air-blowing device) that full husk is separated with empty husk.After separation, use commercially available counter that two batches of seeds are all counted then.Discard the sky husk.On analytical balance, take by weighing the weight of full husk, use digital picture to measure the cross-sectional area of seed.This method produces with next group seed relevant parameter set.

Every paniculiform flower is estimated the average number (coming divided by the first paniculiform number derivation from total number seeds) of every paniculiform Xiao Hua on the plant.The highest panicle be regarded as first panicle with the highest all panicles of panicle eclipsed when the vertical comparison and count by manual.By the number of counting remaining full husk behind separating step, determine the number of full seed.Measure total seed production (seed gross weight) by taking by weighing from the weight of all full husks of plant results.Measure total number seeds of every plant by counting from the number of the husk of plant results, it is corresponding to the number of every strain plant Xiao Hua.Calculate thousand seed weight (TKW) from the number of full seed and its gross weight of counting.Harvest index is defined as seed gross weight and ground area (mm ²) between ratio multiply by the factor 10 ⁶Parameter EmerVigor indication growth of seedling gesture.According to the area that covers by the leaf biomass in the imaging first time (with mm ²Expression) calculates.The plumpness of the full rate of seed (fillrate) indication seed.Its number that is expressed as full seed accounts for the ratio (representing with %) of Xiao Hua number (seed sum).

Use image analysis software to derive these parameters from digital picture, then these parameters are carried out statistical analysis in the mode of automatization.Use the parameter (comprising width, length, area, weight) of measuring single seed with image analysis software link coupled customizing device (by two primary clusterings, weigh and imaging device, form).

Embodiment 9: the measurement of the output correlation parameter of the pGOS2::SYR transformant of growing under the normal growth condition:

Based on above-mentioned seed analysis, the present inventor find with PGOS2::SYR gene construct plant transformed with lack the genetically modified plant of SYR and compare and have higher seed production (being expressed as the number of full seed, the gross weight and the harvest index of seed).The p value shows that this increase is significant.The method that is used for statistical study is the method that the Introductory part at embodiment provides.

The result who obtains for the plant of T1 in generation is summarized among the table E, and described result is subjected to the mean value of test product system for all:

Table E:

	% difference	The P value
	% difference	The P value	The number of full seed	+47	0.0000
The seed gross weight	+52	0.0000	The number of full seed	+47	0.0000
The seed gross weight	+52	0.0000	Harvest index	+54	0.0000

The data that obtain at SYR in first experiment in second experiment of using the T2 plant, have been confirmed.Selection has 4 strains of correct expression pattern and further analyzes.By the expression of monitoring mark, screening is from the seed lot (Seed batch) of the sun plant among the T1 (heterozygote and homozygote).For the incident of each selection, keep the heterozygote seed lot then to carry out the T2 assessment.In every batch of seed, the positive of growth equal amts and heliophobous plant are to assess in the greenhouse.Compare with lacking the genetically modified plant of SYR, the measurement of seed production parameter shows the increase of number, seed gross weight and the harvest index of full seed once more.

Embodiment 10: the measurement of the output correlation parameter of the pGOS2::SYR transformant of growing under stress conditions:

Analysis based on above-mentioned seed, the inventor finds to compare with lacking the genetically modified plant of SYR, with the conversion of pGOS2::SYR gene construct and under salt stress growing plants have higher seed production (being expressed as the number of full seed, the gross weight of seed, full rate and harvest index).In addition, these are compared with control plant by the plant of salt stress to have higher growth of seedling gesture.When growing plant under drought stress, transgenic plant are compared with the genetically modified plant of shortage SYR has the higher seed gross weight and the harvest index of increase.These differences are significant, and the P value of checking from F is lower than 0.05.

The measurement of the output correlation parameter of embodiment 11:pHMGP::SYR transformant:

Similarly in pGOS2::SYR gene construct plant transformed, the present inventor find with pHMGP::SYR gene construct plant transformed with lack the genetically modified plant of SYR and compare and have higher seed production (being expressed as the number of full seed, the gross weight and the harvest index of seed).The P value shows that this increase is significant.

The T1 that obtains is summarized in table F for the result of plant, and described result is subjected to the mean value of test product system for all:

Table F:

	% difference	The p value
	% difference	The p value	The number of full seed	+34	0.0000
The seed gross weight	+33	0.0000	The number of full seed	+34	0.0000
The seed gross weight	+33	0.0000	Harvest index	+37	0.0000

Embodiment B: FG-GAP

Embodiment 12: the evaluation of the sequence relevant with SEQ ID NO:46 with SEQ ID NO:45

Use the database sequence research tool, for example basic local comparison instrument (BLAST) (people (1990) J.Mol.Biol.215:403-410 such as Altschul; With people (1997) NucleicAcids Res.25:3389-3402 such as Altschul), in the sequence of the Entrez of American National biotechnology information center (NCBI) Nucleotide database maintenance, identify sequence (full-length cDNA, EST or genome sequence) relevant and/or the protein sequence relevant with SEQ ID NO:46 with SEQ ID NO:45.Use blast program, by nucleic acid or peptide sequence are compared with sequence library, and by calculating the significance,statistical of coupling, to have the zone of local similar between the searching sequence.To SEQID NO:45 encoded polypeptide utilization TBLASTN algorithm, use default setting, open strainer, to ignore the low complex degree sequence.The output form of analyzing is for comparing in twos, and sorts according to probability score (E value), and wherein score value reflects the occurrent probability of specific comparison (the E value is low more, and the significance of hit event is high more).Except the E value, also keep the score to relatively carrying out identity per-cent.Identity per-cent is meant the number of the identical Nucleotide (or amino acid) on length-specific between two nucleic acid (or polypeptide) sequences that compare.In some cases, can adjust the strict degree of default parameter with the change search.

Except the public obtainable nucleotide sequence that can on NCBI, obtain, also can search for other sequence libraries according to the method identical with aforesaid method.

The nucleic acid that table G provides the nucleotide sequence represented with SEQ ID NO:45 and the protein sequence represented with SEQ ID NO:46 is relevant and the tabulation of protein sequence.

Table G: the corresponding polypeptide that can be used for nucleotide sequence the inventive method, relevant with nucleotide sequence (SEQ ID NO:45) and derive.

Title	The source is biological	Nucleic acid SEQID NO:	Polypeptide SEQID NO:	Database login number	State
Title	The source is biological	Nucleic acid SEQID NO:	Polypeptide SEQID NO:	Database login number	State	AtFG-GAP	Arabidopis thaliana	45	46		Total length
The AtFG-GAP homologue	Arabidopis thaliana	54	55	NM_114965	Total length	AtFG-GAP	Arabidopis thaliana	45	46		Total length
The AtFG-GAP homologue	Arabidopis thaliana	54	55	NM_114965	Total length	OsFG-GAP homologue 1	Rice	56	57	NM_185137	Total length
OsFG-GAP homologue 2	Rice	58	59	AK068943	Total length	OsFG-GAP homologue 1	Rice	56	57	NM_185137	Total length
OsFG-GAP homologue 2	Rice	58	59	AK068943	Total length	The TaFG-GAP homologue	Common wheat	60	/	CK207217	Part
Zm FG-GAP homologue	Zea mays		61	/	AY111316	The TaFG-GAP homologue	Common wheat	60	/	CK207217	Part	Part
Zm FG-GAP homologue	Zea mays		61	/	AY111316	The StFG-GAP homologue	Potato		62	/	BG598275	Part	Part
The AFG-GAP homologue	Aquilegia		63	/	DT735817	The StFG-GAP homologue	Potato		62	/	BG598275	Part	Part
The AFG-GAP homologue	Aquilegia		63	/	DT735817	The BnFG-GAP homologue	Colea	64	/	CX192752	Part	Part
The CsFG-GAP homologue	Sweet orange		65	/	CX674859	The BnFG-GAP homologue	Colea	64	/	CX192752	Part	Part
The CsFG-GAP homologue	Sweet orange		65	/	CX674859	The AoFG-GAP homologue	Officinalis	66	/	CV288972	Part	Part
PFG-GAP homologue 1	Populus	67	/	CN520999	Part	The AoFG-GAP homologue	Officinalis	66	/	CV288972	Part
PFG-GAP homologue 1	Populus	67	/	CN520999	Part	PFG-GAP homologue 2	Populus	68	/	CX176799	Part

The EeFG-GAP homologue	The breast oar root of Beijing euphorbia	69	/	DV130386	Part
The EeFG-GAP homologue	The breast oar root of Beijing euphorbia	69	/	DV130386	Part	The CrFG-GAP homologue	Ceratopterisrichardii		70	/	CV736049	Part
The WmFG-GAP homologue	Orchid at the age of one hundred years old	71	/	DT601669	Part	The CrFG-GAP homologue	Ceratopterisrichardii		70	/	CV736049	Part
The WmFG-GAP homologue	Orchid at the age of one hundred years old	71	/	DT601669	Part	The MsFG-GAP homologue	Alfalfa	72	SEQ ID NO:73 to SEQ ID NO:76		Part

Embodiment 13: the comparison of related polypeptide sequence

From the AlignX of Vector NTI (Invitrogen) based on the clustering algorithm of the progressive comparison of generally using (people (1997) Nucleic Acids Res 25:4876-4882 such as Thompson; People such as Chenna (2003) .Nucleic Acids Res 31:3497-3500).Can use in abutting connection with the clustering algorithm constructing system and set.The default value of the open point penalty in room is 10, and the default value that point penalty is extended in the room is 0.1, and the weight matrix of selection is Blosum 62 (if comparison polypeptide).

Can be used for implementing in the polypeptide of the inventive method in evaluation, what the multiple sequence that uses related polypeptide to carry out was compared the results are shown among Fig. 7.Can be clear that,, on the major part of protein sequence, have sequence conservation although there are some rooms in the comparison.

Embodiment 14: can be used for implementing the calculating of the overall per-cent identity between the peptide sequence of the inventive method

A method MatGAT in the method that use can obtain in this area (matrix overall comparison instrument) software (BMC Bioinformatics.2003 4:29.MatGAT:an application thatgenerates similarity/identity matrices using protein or DNA sequences.Campanella JJ, Bitincka L, Smalley J; Software by Ledion Bitincka trustship), determine to be used to implement overall similarity and identity per-cent between the full-length polypeptide sequence of method of the present invention.MatGAT software need not data are compared in advance, can produce the similarity/identity matrix of DNA or protein sequence.This program is utilized Myers and Miller overall comparison algorithm, and (the open point penalty in room is 12, and to extend point penalty be 2 in the room) carry out a series of comparison in twos, utilize for example Blosum 62 (for polypeptide) calculating similarity and identity, then the result is arranged in distance matrix.Sequence similarity is shown in the diagonal lines Lower Half, and sequence identity is shown in the diagonal lines first half.

More used parameter has:

Matrix: Blosum 62 keeps the score

First room: 12

Extend the room: 2

The overall similarity of peptide sequence length range (the part of polypeptide sequence is foreclosed) and the software analysis of identity the results are shown in table H.The diagonal lines top provides identity per-cent, and the diagonal lines below provides similarity per-cent.

Compare with SEQ ID NO:46, the per-cent identity that can be used for implementing between the peptide sequence of the inventive method can be for being low to moderate 17% amino acid identity.

Table H: the overall similarity in the full-length polypeptide sequence scope and the MatGAT result of identity.

	1	2	3	4
	1	2	3	4	1.AtFGAP1		18.1	65.5	17.4
2.AtFGGAP2	31.4		17.9	67.7	1.AtFGAP1		18.1	65.5	17.4
2.AtFGGAP2	31.4		17.9	67.7	3.OsFGGAP1	76.7	33.5		16.9
4.OsFGGAP2	32.8	83.6	33		3.OsFGGAP1	76.7	33.5		16.9

Embodiment 15: can be used for implementing the evaluation of the structural domain that comprises in the peptide sequence of the inventive method

Protein families, structural domain and site (Integrated Resource of ProteinFamilies, Domains and Sites (the InterPro)) database of reallocating resources is the integrated interface that carries out based on the search of text and sequence tag database commonly used.The InterPro database gets up these database combination, and they utilize diverse ways to learn and the relevant proteinic bioinformation in various degree that fully characterizes, to obtain protein tag.The cooperation database comprises SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs.Interpro is by European information biology institute (the European Bioinformatics Institute) trustship that is positioned at Britain.

The InterPro scanning result of peptide sequence is shown in Table I shown in the SEQ ID NO:46.

Table I: the InterPro scanning result of peptide sequence shown in the SEQ ID NO:46

Database	Accession number	Logon name
Database	Accession number	Logon name	Pfam	PF01839	FG-GAP
INTERPRO	IPR013517	FG-GAP	Pfam	PF01839	FG-GAP
INTERPRO	IPR013517	FG-GAP	INTERPRO	IPR000413	The beta 2 integrin alpha chain

Embodiment 16: be used to implement the topology prediction of the peptide sequence of method of the present invention

TargetP 1.1 can predict the Subcellular Localization of eukaryotic protein.According to this program, the predictability existence based on following arbitrary N-end presequence positions definite: chloroplast transit peptides (cTP), Mitochondrially targeted peptide (mTP) or Secretory Pathway signal peptide (SP).Final forecasting institute based on score value be not real probability, and add up and needn't be 1.But, according to TargetP, the location that score is the highest is most probable, and the relation between the score value (reliability class) can be used as the index of described forecasting reliability.Reliability class (RC) scope from 1 to 5, the wherein the strongest prediction of 1 expression.TargetP is by the server maintenance of Technical University Of Denmark.

For the sequence that contains N-end presequence through prediction, also can predict the potential cleavage site.

TargetP 1.1 analytical resultss of peptide sequence shown in the SEQ ID NO:46 are shown among the following table J.What select is " plant " organism type, does not stipulate cutoff value, and requires the prediction length of transit peptides.The Subcellular Localization of peptide sequence may be in cell shown in the SEQ ID NO:46, existence is to the slight deflection of Secretory Pathway (though have 5 reliability score value), the prediction length of the transit peptides of inferring is 24 amino acid (it is reliable to be not so good as Subcellular Localization prediction itself, has the length variations of several amino acid) that start from N-terminal.

The TargetP 1.1 of peptide sequence analyzes shown in the table J:SEQ ID NO:46

Length (AA)	896
Length (AA)	896	Chloroplast transit peptides	0.010
The mitochondrial transport peptide	0.546	Chloroplast transit peptides	0.010
The mitochondrial transport peptide	0.546	The Secretory Pathway signal peptide	0.643
Other ubcellular targets	0.038	The Secretory Pathway signal peptide	0.643
Other ubcellular targets	0.038	The location of prediction	Secretion
Reliability class		The location of prediction	Secretion
Reliability class		5
The transit peptides length of prediction		5	24

When analyzing, (0.998 probability) positive identification is to existing the N with 24 amino acid lengths to hold secreting signal peptide reliably when using SignalP people such as (, J.Mol.Biol., 340:783-795,2004) Bendtsen.In addition, when using THMM algorithm (Center for Biological SequenceAnalysis, Technical University of Denmark) time, predicts that this protein is positioned at the outside of cell, have only C end afterbody to be present in the tenuigenin: residue 1-859: the outside; Residue 860-879: membrane spaning domain, residue 880-896: inboard.

Can utilize many other algorithms to carry out such analysis, comprise:

● ChloroP 1.1, by Technical University Of Denmark's trust server;

● protein is sought Subcellular Localization forecasting software 1.2 editions, by the trust server of molecular biosciences institute of Brisbane ,Australia University of Queensland;

● PENCE Proteome Analyst PA-GOSUB 2.5, by the trust server of Canada big Alberta Edmonton Alberta university;

Embodiment 17: gene clone

(Invitrogen, Paisley UK) pass through pcr amplification Arabidopis thaliana FG-GAP gene as template to use Arabidopis thaliana seedling cDNA library.Behind the RNA that reverse transcription is extracted from seedling, cDNA is cloned into pCMV Sport6.0.The average inset size in library is 1.5kb, and clone's original number is about 1.59 x 10 ⁷Cfu.At 6 x 10 ¹¹After the amplification first time of cfu/ml, initial titer is confirmed as 9.6 x 10 ⁵Cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.Primer prm06643 (the SEQID NO:47 that will comprise the AttB site that is used for the Gateway reorganization; 5 '-ggggacaagtttgtacaaaaaagcaggcttaaacaatgaaatctcgagcgagg-3 ') and prm06644 (SEQ ID NO:48 justice is arranged, and initiator codon indicates with runic, and the AttB1 site indicates with italics:; Oppositely, complementation; The AttB2 site indicates with italics: 5 '-ggggaccactttgtacaagaaagctgggtcctg tttacagatggtacctagt-3 ') be used for pcr amplification.Under standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.Also use standard method amplification and the purifying 3.2kb PCR fragment of (comprising the attB site).Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, and " clone crosses the threshold " pFG-GAP.Plasmid pDONR201 conduct

The part of technology is available from Invitrogen.

Embodiment 18: the structure of carrier

Clone FG-GAP and the pGOS2 (being used for the purpose carrier that rice transforms) one that will cross the threshold subsequently is used from the LR reaction.This carrier form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and be intended to and the aim sequence that has been cloned into the clone that crosses the threshold carries out the Gateway box of recombinating the LR body in.The rice GOS2 promotor (Nucleotide 1 to 2193 of SEQ IDNO:49, promotor-assortment of genes) that is used for constitutive expression is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, it is LBA4044 that the expression vector pGOS2::FG-GAP (Fig. 7) that is used for FG-GAP of gained is transformed into the Agrobacterium strain, and then is transformed into rice plant.Make rice plant's growth of conversion, with regard to the parameter of describing among the embodiment 19 it is checked then.

About the information of other farm crop referring to embodiment 40.

Embodiment 19: to using the appraisal procedure of the FG-GAP plant transformed under the control of rice GOS2 promotor

Produce about 15 to 20 T0 rice transformant independently.With former generation transformant be transferred to from tissue culture room and grow the greenhouse and gather in the crops the T1 seed.Isolating 7 incidents of 3:1 take place with regard to genetically modified existence/with regard to not existing in reservation in the T1 offspring.For each incident in these incidents,, select about 10 strains to comprise the T1 seedling of transgenosis (heterozygote and homozygote) and the T1 seedling that about 10 strains lack transgenosis (invalid zygote) by the expression of monitoring witness marking.The T1 plant of selecting is transferred to the greenhouse.Every strain plant accepts to make clearly phenotypic data and the corresponding related unique bar code mark of plant.The T1 plant selected is provided with at following environment is grown in down in the soil of flowerpot that diameter is 10cm, described environment setting is: photoperiod=11.5h, intensity of illumination=30,000lux or stronger, daytime temperature=28 ℃ or higher, nocturnal temperature=22 ℃, relative humidity=60-70%.Transgenic plant and corresponding invalid zygote are grown at random position side by side.From sowing time to the ripening stage, plant is passed through the digital imagery chamber for several times.On each time point, to every strain plant from least 6 different angle shot digital pictures (2048 x, 1536 pixels, 1,000 6 hundred ten thousand pigments).

Determine plant area (or leaf biomass) on the ground by the sum that is different from the pixel of background on the digital picture of counting plant part on the ground.With regard to the image of taking from different perspectives on the identical time point this value is averaged, convert described value to represent physical surface value by calibration then with square millimeter.Experiment shows that the ground plant area of measuring by this method is related with the biomass of ground plant part.Areamax is the ground area when plant reaches its maximum leaf biomass.

Gather in the crops sophisticated main panicle, packed, with barcode it is carried out mark, under 37 ℃ in baking oven dry 3 days then.To the panicle threshing, collect all seeds then.Use gas blower that full husk is separated with empty husk.After separation, use commercially available counter that two batches of seeds are all counted then.Discard the sky husk.On analytical balance, take by weighing the weight of full husk, use digital picture to measure the cross-sectional area of seed.This method produces one group of following seed correlation parameter:

Every paniculiform number of spending is for estimating the parameter of the average number of every paniculiform Xiao Hua on the plant, and it can get divided by the first paniculiform number from the seed overall number.The highest panicle be regarded as first panicle with the highest all panicles of panicle eclipsed when the vertical comparison and count by manual.By counting the number of the full husk that behind separating step, stays, determine the number of full seed.Measure total seed production (seed gross weight) by taking by weighing from the weight of all full husks of plant results.Measure total number seeds of every strain plant by counting from the number of the husk of plant results, it is corresponding to the number of the Xiao Hua of every strain plant.Number and its gross weight according to the full seed of counting are calculated thousand seed weight (TKW).Harvest index is defined as seed gross weight and ground area (mm ²) between ratio multiply by the factor 10 ⁶Can use image analysis software to derive these parameters from digital picture, then these parameters be carried out statistical analysis in the mode of automatization.Use and image analysis software link coupled customizing device (by 2 primary clusterings, weigh and imaging device, form), measure the parameter (comprising width, length, area, weight) of single seed.

With regard to imbalance design and gauged dual factors ANOVA (variance analysis) as the statistical models of net assessment plant phenotype feature.All measuring parameters with all incidents of all plants of this gene transformation are carried out the F check.Carry out the F check with the influence of checking all transformation events of gene pairs and the population effect of verifying gene, be also referred to as " overall genetic effect " herein.If the value display data of F check is significant, then reach a conclusion---there is " gene " effect, this means that what cause this effect is not only the existence or the position of gene.For this F check, the significance threshold setting of real overall genetic effect is on 5% probability level.

For the intragentic effect of inspection incident, promptly check the strain specificity effect, use data set from transgenic plant and corresponding invalid plant, in each incident, implement the t check." invalid plant " or " invalid segregant " or " invalid zygote " are meant the plant of handling in the mode identical with transgenic plant, but transgenosis is separated from this plant.The feminine gender that invalid plant also can be described to isozygoty transforms plant.With the significance threshold setting of T check on 10% probability level.The possibility of result of some incidents is higher or lower than this threshold value.This is based on hypothesis: gene may only have effect on genomic some position, and this position dependence effect is not rare.The genetic effect of the type is also referred to as " the strain effect of gene " herein.By with the t value and t-distribution compares or selectively by F value and F-distribution are compared, obtain the p value.To provide null hypothesis (promptly not having the transgenosis effect) be correct probability to the p value then.

The data that obtain at FG-GAP in first experiment in second experiment of using the T2 plant, have been confirmed.Select 4 strains further to analyze.By the expression of monitoring mark, screening is from the seed lot of the sun plant among the T1 (heterozygote and homozygote).For the incident of each selection, keep the heterozygote seed lot then to carry out the T2 assessment.In every batch of seed, the positive of growth equal amts and heliophobous plant are to assess in the greenhouse.

In T2 generation, assessed the FG-GAP that adds up to 120 strains and transformed plant, promptly every incident 30 strain plants, wherein 15 strains be transgenosis because of male, 15 strains are the transgenosis feminine gender.

Because two experiments carrying out have overlapping events, therefore carry out combinatory analysis.This can be used for checking the consistence of effect in two experiments, and if situation if this is really true, thereby it can be used for collecting evidence from two experiments and increases the credibility of conclusion.The method of using is to consider the method with mixed model of the multilevel hierarchy of data (i.e. experiment-incident-segregant).By the distribution of the side of card is compared with likelihood ratio test, obtain the p value.

The assessment of embodiment 20:FG-GAP transformant: the measurement of output correlation parameter

Based on the analysis of above-mentioned seed, the present inventor finds to compare and have higher seed production with lacking the genetically modified plant of FG-GAP with FG-GAP gene construct plant transformed, and described seed production shows as the number and the seed gross weight of full seed.The P value shows that this increase is significant.Harvest index also increases (+9%) in addition.

The T1 that obtains is summarized among the table K for the result of plant:

Table K:

	% difference	The p value of F check
	% difference	The p value of F check	The number of full seed	+19	0.0051
The seed gross weight	+17	0.0199	The number of full seed	+19	0.0051

Obtain these positive findingses at T2 in generation once more.In table L, the overall percentage of data presentation full seed, seed gross weight and harvest index increases (according to the data computation of each single line in T2 generation) and P value accordingly.The result in associating T1 generation these T2 data of reappraising in combinatory analysis, the observed effect of P value demonstration of acquisition is a highly significant.

Table L:

Embodiment C: CYP90B

Embodiment 21: the gene clone of rice CYP90B cDNA

(Invitrogen, Paisley UK) pass through pcr amplification rice CYP90B cDNA as template to use rice seedling cDNA library.Behind the RNA that reverse transcription is extracted from seedling, cDNA is cloned into pCMV Sport6.0.The average inset size in library is 1.6kb, and clone's original number is about 1.67 x 10 ⁶Cfu.At 6 x 10 ¹⁰After the amplification first time of cfu/ml, initial titer is defined as 3.34x 10 ⁶Cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.Primer (the SEQ ID NO:107 that will comprise the AttB site that is used for the Gateway reorganization; 5 ' GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGGCCGCCATGATGGC3 ') and (SEQ ID NO:108 justice is arranged, and initiator codon indicates with runic, and the AttB1 site indicates with italic:; Oppositely, complementation; The AttB2 site indicates with italic: 5 ' GGGGACCACTTTGTACAAGAAAGCTGGGT TTACTCCTGCTCATCATCC3 ') be used for pcr amplification.Under standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.Also use standard method amplification and purifying 1585bp (to comprise the attB site; From initial to stopping 1521bp) the PCR fragment.Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, " clone crosses the threshold ".Plasmid pDONR201 is as Gateway

The part of technology is available from Invitrogen.

Embodiment 22: the structure of carrier

The clone that will cross the threshold subsequently is used from the LR reaction with the purpose carrier one that is used for the rice conversion.These carriers form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and be intended to be used for carry out the Gateway box of recombinating the LR body in the aim sequence that has been cloned into the clone that crosses the threshold.Use is positioned at 4 different rice promoters of this Gateway box upstream and expresses rice CYP90B: prolamine RP6, oleosin 18kDa, GOS2 and HMGB1.

After the LR reconstitution steps, it is LBA4044 that the expression vector (prolamine RP6 promotor, oleosin 18kDa, GOS2 and HMGB1-are referring to Figure 14) of gained is transformed into the Agrobacterium strain, is transformed into rice plant then.Make rice plant's growth of conversion, with regard to the parameter of describing in the following example it is checked then.About the conversions of other farm crop referring to example example 40.

Embodiment 23: the description of phenotype appraisal procedure

Every construct produces about 15 to 20 T0 rice transformant independently.With former generation transformant be transferred to the greenhouse to grow and to gather in the crops the T1 seed from tissue culture room.Reservation is with regard to genetically modified existence/the T1 offspring does not present 4 incidents in isolating 5 incidents of 3:1 with regard to not existing.For each incident in these incidents,, select about 10 strains to comprise the T1 seedling of transgenosis (heterozygote and homozygote) and the T1 seedling that about 10 strains lack transgenosis (invalid zygote) by the expression of monitoring witness marking.Transgenic plant and suitable control plant are grown at random position side by side.From sowing time to the ripening stage, plant is passed through the digital imagery chamber for several times.On each time point, to each strain plant from least 6 different angle shot digital pictures (2048 x, 1536 pixels, 1,000 6 hundred ten thousand pigments).

According to the appraisal procedure identical (but the assessment of every incident is more individual) with T1 generation in T2 further 3 T1 incidents of assessment in generation.

The measurement of seed correlation parameter

Gather in the crops sophisticated main panicle, counting, packed, use bar code label, under 37 ℃ in baking oven dry 3 days then.Then to the panicle threshing, collect all seeds and count.Use gas blower that full husk is separated with empty husk.Discard the sky husk, then the residue fraction is counted once more.On analytical balance, take by weighing the weight of full husk.Determine the number of full seed by the number of counting the full husk that behind separating step, stays.Measure total seed production by taking by weighing from the weight of all full husks of plant results.Measure total number seeds of every strain plant by counting from the number of the husk of plant results.Number and its gross weight according to the full seed of counting are calculated thousand seed weight (TKW).Harvest index among the present invention (HI) is defined as seed ultimate production and ground area (mm ²) between ratio multiply by the factor 106.The sum of the every paniculiform flower that defines among the present invention is the ratio between seed sum and the paniculiform number of sophisticated master.The full rate of the seed that defines among the present invention is the ratio (representing with %) of the number of the full seed sum that accounts for seed (or Xiao Hua).Use the parameter (width, length and area) of measuring single seed with image analysis software link coupled customizing device, described device is made up of 2 primary clusterings (weighing and imaging device).The seed with shelling of band shell all is used for these measurements.

Statistical analysis: F check

With the statistical models of dual factors ANOVA (variance analysis) as net assessment plant phenotype feature.All measuring parameters with all incidents of all plant of gene transformation of the present invention are carried out the F check.Carry out the effect of F check with all transformation events of inspection gene pairs, and the population effect of check gene, also be called " overall genetic effect ".The significance threshold value of real overall genetic effect is set to 5% probability level of F check.If significance F test value points to certain genetic effect, this means to be not only that the existence of gene or position cause difference on the phenotype.

Embodiment 24: the result of the rice CYP90B under non-constitutive promoter control

24.1 under endosperm specificity promoter control, express the transgenic plant of CYP90B

Seed production and the HI measuring result of expressing the transgenic plant of CYP90B under the control of endosperm-specific (prolamine RP6) promotor are shown in respectively among table M and the N.Shown the number of incident and from the p value of the F check in T1 and T2 generation with increase.

Table M: the seed production measuring result of under endosperm specificity promoter control, expressing the transgenic plant of CYP90B.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 43	11	0.1572
T2 generation	In 33	13	0.0103	T1 generation	In 43	11	0.1572

Table N: the HI measuring result of under endosperm specificity promoter control, expressing the transgenic plant of CYP90B.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 44	11	0.047
T2 generation	In 33	10	0.0392	T1 generation	In 44	11	0.047

When comparing with control plant, although the ground phytomass remains unchanged (data not shown), but because the increase of seed production, the transgenosis rice plant of expressing CYP90B under the control of endosperm-specific (prolamine RP6) promotor presents the results of increase.

24.2 under embryo/aleuron specificity promoter control, express the transgenic plant of CYP90B

The TKW measuring result of expressing the transgenic plant of CYP90B under the control of embryo/aleuron (oleosin 18kDa) promotor is shown among the table O.Shown the number of incident and from the p value of the F check in T1 and T2 generation with increase.

Table O: the TKW measuring result of under embryo/aleuron promotor control, expressing the transgenic plant of CYP90B.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 55	4	0.0002
T2 generation	In 32	1	0.2428	T1 generation	In 55	4	0.0002

The average seed area measurement of expressing the transgenic plant of CYP90B under the control of oleosin 18kDa promotor the results are shown among the table P.Shown the number of incident and from the p value of the F check in T1 and T2 generation with increase.

Table P: the average seed area measurement result who under embryo/aleuron promotor control, expresses the transgenic plant of CYP90B.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 55	3	<0.0001
T2 generation	In 33	2	0.0272	T1 generation	In 55	3	<0.0001

The average seed length measuring result of expressing the transgenic plant of CYP90B under the control of oleosin 18kDa promotor is shown among the table Q.Shown the number of incident and from the p value of the F check in T1 and T2 generation with increase.

Table Q: the average seed length measuring result of under embryo/aleuron promotor control, expressing the transgenic plant of CYP90B.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 55	3	<0.0001
T2 generation	In 33	1	0.0086	T1 generation	In 55	3	<0.0001

The transgenosis rice plant of expressing CYP90B under the control of embryo/aleuron (oleosin 18kDa) promotor has the seed that increase takes place for TKW, seed area and seed length.Do not observe the remarkable increase of seed production.

Embodiment 25: assessment and the result of the rice CYP90B under constitutive promoter control

25.1 under the control of GOS2 constitutive promoter, express the transgenic plant of CYP90B

The assessment of the measurement result of expressing the transgenic plant of CYP90B under the control of GOS2 constitutive promoter is shown among the table R.Shown the number of incident and from the p value of the F check in T1 and T2 generation with increase.When T1 obtains negative findings in generation, do not carry out the T2 assessment in generation.

Table R: the assessment of the measurement result of under the control of GOS2 constitutive promoter, expressing the transgenic plant of CYP90B

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	Ground biomass	In 55	-13	<0.0001
Highly	In 55	-7	<0.0001	Ground biomass	In 55	-13	<0.0001
Highly	In 55	-7	<0.0001	The number of full seed	In 55	-53	<0.0001
The number of seed	In 55	-32	<0.0001	The number of full seed	In 55	-53	<0.0001
The number of seed	In 55	-32	<0.0001	Seed production	In 55	-53	<0.0001
HI	In 55	-46	<0.0001	Seed production	In 55	-53	<0.0001

25.2 under the control of HMBG1 constitutive promoter, express the transgenic plant of CYP90B

The assessment of the measurement result of expressing the transgenic plant of CYP90B under the control of HMGB1 constitutive promoter is shown among the table S.Shown the number of incident and from the p value of the F check in T1 generation with increase.When T1 obtains negative findings in generation, do not carry out the T2 assessment in generation.

Table S: the assessment of the measurement result of under the control of HMGB1 constitutive promoter, expressing the transgenic plant of CYP90B.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	Ground biomass	In 55	-18	<0.0001
Highly	In 55	-6	<0.0001	Ground biomass	In 55	-18	<0.0001
Highly	In 55	-6	<0.0001	The number of full seed	In 55	-56	<0.0001
The number of seed	In 55	-33	<0.0001	The number of full seed	In 55	-56	<0.0001
The number of seed	In 55	-33	<0.0001	Seed production	In 55	-56	<0.0001
HI	In 55	-46	<0.0001	Seed production	In 55	-56	<0.0001

Compare with control plant, the transgenic plant of expressing CYP90B under 2 kinds of different constitutive promoter controls demonstrate number, seed production and the HI of the ground phytomass of strong minimizing, plant height, full seed.

Embodiment D:CDC27

Embodiment 26: the NH that is coded in polypeptide ₂End regions has the clone of arabidopsis gene of CDC27 polypeptide of the TPR structural domain of at least one inactivation

(UK) as template, by the arabidopsis gene of pcr amplification coding CDC27 polypeptide, described CDC27 polypeptide (CDS0171_2) is at the NH of polypeptide for Invitrogen, Paisley to use Arabidopis thaliana seedling cDNA library ₂End regions has the TPR structural domain of at least one inactivation.Behind the RNA that reverse transcription is extracted from seedling, cDNA is cloned into pCMV Sport 6.0.The average inset size in library is 1.5kb, and clone's original number is about 1.59 x 10 ⁷Cfu.10 ¹⁰After the amplification first time of cfu/ml, initial titer is defined as 9.6 x 10 ⁵Cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.Primer (the SEQ ID NO:149 that will comprise the AttB site that is used for the Gateway reorganization; 5 '-GGGGACAAGTTTGTACAAAAAAGCA GGCTTCACAATGCAACAACTGTCAACTTC3 ') and (SEQ ID NO:150 justice is arranged, and initiator codon indicates with runic, and the AttB1 site indicates with italics:; Oppositely, complementation; The AttB2 site indicates with italics: 5 ' GGGGACCACTTTGTACAAGAAAGCTGGGTTGGAGTAGCTATGGTTTCAC-3 ') be used for pcr amplification.Under standard conditions, use Hifi TaqDNA polysaccharase to carry out PCR.Also use standard method amplification and purifying 1816bp (to comprise the attB site; From initial to stopping 1737bp) the PCR fragment.Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, " clone crosses the threshold ".Plasmid pDONR201 is as Gateway

The part of technology is available from Invitrogen.

Embodiment 27: the structure of carrier

The clone that will cross the threshold subsequently is used from the LR reaction with the purpose carrier one that is used for the rice conversion.This carrier form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and be intended to be used for carry out the Gateway box of recombinating the LR body in the aim sequence that has been cloned into the clone that crosses the threshold.The rice OSH1 promotor (SEQ ID NO:151) that is used for the expression of stem end meristematic tissue is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, it is LBA4044 that the expression vector of the gained shown in Figure 18 is transformed into the Agrobacterium strain, and then is transformed into rice plant.Make rice plant's growth of conversion, with regard to the parameter of describing in embodiment 28 and 29 it is checked then.About the conversions of other farm crop referring to embodiment 40.

Embodiment 28: the description of phenotype appraisal procedure

Produce about 15 to 20 T0 rice transformant independently.With former generation transformant be transferred to the greenhouse to grow and to gather in the crops the T1 seed from tissue culture room.Keep the T1 offspring with regard to genetically modified existence/present with regard to not existing isolating 5 incidents of 3:1.For each incident in these incidents,, select about 10 strains to comprise the T1 seedling of transgenosis (heterozygote and homozygote) and the T1 seedling that about 10 strains lack transgenosis (invalid zygote) by the expression of monitoring witness marking.Transgenic plant and suitable control plant are grown at random position side by side.From sowing time to the ripening stage, plant is passed through the digital imagery chamber for several times.On each time point, to every strain plant from least 6 different angle shot digital pictures (2048 x, 1536 pixels, 1,000 6 hundred ten thousand pigments).

According to the appraisal procedure identical (but the assessment of every incident is more individual) with T1 generation in T2 further 3 incidents of in T1, assessing of assessment in generation.

The measurement of seed correlation parameter

Gather in the crops sophisticated main panicle, counting, packed, use bar code label, under 37 ℃ in baking oven dry 3 days then.To the panicle threshing, collect all seeds and count then.Use gas blower that full husk is separated with empty husk.Discard the sky husk, then the residue fraction is counted once more.On analytical balance, take by weighing the weight of full husk.Determine the number of full seed by the number of counting the full husk that behind separating step, stays.Measure total seed production by taking by weighing from the weight of all full husks of plant results.Measure total number seeds of every strain plant by counting from the number of the husk of plant results.Number and its gross weight according to the full seed of counting are calculated thousand seed weight (TKW).Harvest index of the present invention (HI) is defined as seed gross weight and ground area (mm ²) between ratio multiply by the factor 10 ⁶The sum of the every paniculiform flower that defines among the present invention is the ratio between seed sum and the paniculiform number of sophisticated master.The full rate of the seed that defines among the present invention is the ratio (representing with %) that the number of full seed accounts for seed (or Xiao Hua) sum.

Statistical analysis: F check

Embodiment 29: the assessment result of expressing the transgenosis rice plant of modified Arabidopis thaliana CDC27 nucleic acid under the control of stem end meristematic tissue promotor

The assessment of the measurement result (number of seed production, full seed and HI) of expressing the transgenic plant of modified CDC27 nucleic acid under stem end meristematic tissue promotor (OSH1) control is shown among the table T to V.Shown the number of incident with increase, with the % difference of suitable control plant and from the p value T of the F check in T1 and T2 generation.

Table T: the seed production measuring result of under the control of stem end meristematic tissue promotor, expressing the transgenic plant of modified CDC27 nucleic acid.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 53	35	0.0113
T2 generation	In 32	11	0.0083	T1 generation	In 53	35	0.0113

Table U: the measuring result of full seed number of under stem end meristematic tissue promotor control, expressing the transgenic plant of modified CDC27 nucleic acid.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 53	36	0.0083
T2 generation	In 32	10	0.0099	T1 generation	In 53	36	0.0083

Table V: the measuring result of harvest index of under stem end meristematic tissue promotor control, expressing the transgenic plant of modified CDC27 nucleic acid.

	The number that shows the incident that increases	% difference	The P value of F check
	The number that shows the incident that increases	% difference	The P value of F check	T1 generation	In 53	34	0.0053
T2 generation	In 32	6	0.0188	T1 generation	In 53	34	0.0053

The transgenosis rice plant of expressing modified CDC27 nucleic acid under the control of stem end meristematic tissue promotor has the seed production of remarkable increase, the full seed number of increase and the harvest index of increase.

Embodiment E: AT hook

Embodiment 30: the gene clone of the AT crocheting sign indicating number nucleic acid of rice

(Invitrogen, Paisley UK) as template, comprise the rice gene (referring to SEQID NO:152) of the polypeptide of AT hook structure territory and DUF296 structural domain by the pcr amplification coding to use rice seedling cDNA library.Behind the RNA that reverse transcription is extracted from seedling, cDNA is cloned into pCMVSport 6.0.The average inset size in library is 1.6kb, and clone's original number is about 1.67 x 10 ⁷Cfu.At 6 x 10 ¹⁰After the amplification first time of cfu/ml, initial titer is defined as 3.34 x 10 ⁶Cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.Primer (the SEQ ID NO:196 that will comprise the AttB site that is used for the Gateway reorganization; 5 '-ggggacaagtttgtacaaaaaagcaggcttaaacaatggatccggtcacgg-3 ') and (SEQID NO:197 justice is arranged, the AttB1 primer:; Oppositely, complementation; AttB2 primer: 5 '-ggggaccactttgtacaagaaagctgggtggaatcgatccatctcagaa-3 ') be used for pcr amplification.Under standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.Use standard method amplification and purifying PCR fragment (to comprise the attB site; Extremely stop from initial).Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, " clone crosses the threshold ".Plasmid pDONR201 conduct

The part of technology is available from Invitrogen.

Embodiment 31: the structure of carrier

The clone that will cross the threshold subsequently is used from the LR reaction with the purpose carrier one that contains the prolamine promotor that is used for the rice conversion.This carrier form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and be intended to be used for carry out the Gateway box of recombinating the LR body in the aim sequence that has been cloned into the clone that crosses the threshold.The paddy prolamine promotor (SEQ ID NO:195) that is used for endosperm specificity expression is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, it is LBA4044 that the gained expression vector shown in Figure 22 is transformed into the Agrobacterium strain, and then is transformed into rice plant.Make rice plant's growth of conversion, the parameter that just describes below is then checked it.About the conversions of other farm crop referring to embodiment 40.

Embodiment 32: assessment and result

Produce about 15 to 20 T0 rice transformant independently.With former generation transformant be transferred to from tissue culture room and grow the greenhouse and gather in the crops the T1 seed.Keep the T1 offspring with regard to genetically modified existence/present with regard to not existing isolating 7 incidents of 3:1.For each incident in these incidents,, select about 10 strains to comprise the T1 seedling of transgenosis (heterozygote and homozygote) and the T1 seedling that about 10 strains lack transgenosis (invalid zygote) by the expression of monitoring witness marking.

32.1 statistical analysis: F check

32.2 the measurement of seed correlation parameter

Gather in the crops sophisticated main panicle, counting, packed, use bar code label, under 37 ℃ in baking oven dry 3 days then.To the panicle threshing, collect all seeds and count then.Use gas blower that full husk is separated with empty husk.Discard the sky husk, then the residue fraction is counted once more.On analytical balance, take by weighing the weight of full husk.Determine the number of full seed by the number of counting the full husk that behind separating step, stays.Measure total seed production by taking by weighing from the weight of all full husks of plant results.Measure total number seeds of every strain plant by counting from the number of the husk of plant results.Number and its gross weight according to the full seed of counting are calculated thousand seed weight (TKW).Harvest index (HI) is expressed as seed ultimate production and ground area (mm ²) between ratio multiply by the factor 10 ⁶The sum of every paniculiform flower is expressed as the ratio between seed sum and the paniculiform number of sophisticated master.The number that the full rate of seed is expressed as full seed accounts for the total per-cent of seed (or Xiao Hua).

Table W: show and compare, the comparative data of the difference of the seed production that use endosperm specificity promoter (prolamine) obtains with root-specific promoter (RCc3 promotor)

This table shows with corresponding control plant (invalid zygote) compares the percentage difference of transgenic plant on various parameters; P value from the population effect of the demonstration gene of F check also is shown in the table.As shown in the table, be to express in the plant of AT crocheting sign indicating number nucleic acid (SEQID NO:152) under the endosperm specificity promoter control, multiple seed production parameter increases, yet in transgenic plant, express identical genetically modified plant down, acquisition increase (in fact reducing significantly) for being root-specific promoter control.

Embodiment F: DOF transcription factor

Embodiment 33: the gene clone of Arabidopis thaliana DOF transcription factor (SEQ ID NO:198)

(Invitrogen, Paisley UK) pass through pcr amplification Arabidopis thaliana DOF transcription factor gene as template to use Arabidopis thaliana seedling cDNA library.Behind the RNA that reverse transcription is extracted from seedling, cDNA is cloned into pCMV Sport 6.0.The average inset size in library is 1.5kb, and clone's original number is 1.59 x 10 ⁷The rank of cfu.At 6 x 10 ¹¹After the amplification first time of cfu/ml, initial titer is defined as 9.6 x 10 ⁵Cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.5 ' ggggacaagtttgtacaaaaaagcaggcttaaacaatgggtggatcgatggc3 ') and (SEQ ID NO:224) (reverse complemental AttB2 primer: 5 ' ggggaccactttgtacaagaaagctgggtcgttaatgatccgacaaaaca3 ') be used for pcr amplification the primer (SEQ ID NO:223) that will comprise the AttB site that is used for Gateway reorganization (has an adopted AttB1 primer:.In standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.Also use standard method amplification and purifying PCR fragment (to comprise the attB site; Extremely stop from initial).Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, " clone crosses the threshold ".Plasmid pDONR201 conduct

The part of technology is available from Invitrogen.

Embodiment 33a: the structure of carrier

The clone that will cross the threshold subsequently is used from the LR reaction with the purpose carrier one that comprises GOS2 that is used for the rice conversion.This carrier form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and be intended to be used for carry out the Gateway box of recombinating the LR body in the aim sequence that has been cloned into the clone that crosses the threshold.The rice GOS2 promotor (SEQ ID NO:225) that is used for constitutive expression is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, it is LBA4044 that the gained expression vector shown in Figure 26 is transformed into the Agrobacterium strain, and then is transformed into rice plant.Make rice plant's growth of conversion, the parameter that just describes below is then checked it.About the conversions of other farm crop referring to embodiment 40.

Embodiment 34: the gene clone of Arabidopis thaliana DOF transcription factor (SEQ ID NO:226)

(Invitrogen, Paisley UK) pass through pcr amplification Arabidopis thaliana DOF transcription factor gene as template to use Arabidopis thaliana seedling cDNA library.Behind the RNA that reverse transcription is extracted from seedling, cDNA is cloned into pCMV Sport6.0.The average inset size in library is 1.5kb, and clone's original number is 1.59 x 10 ⁷Cfu.At 6 x 10 ¹¹After the amplification first time of cfu/ml, initial titer is defined as 9.6 x 10 ⁵Cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.5 ' ggggacaagtttgtacaaaaaagcaggcttaaacaatgatgatggagactagagat c3 ') and (SEQ ID NO:257) (reverse complemental AttB2 primer: 5 ' ggggaccactttgtacaagaaagctgggtcatatgtaactctaaatctgttca3 ') be used for pcr amplification the primer (SEQ ID NO:256) that will comprise the AttB site that is used for Gateway reorganization (has an adopted AttB1 primer:.In standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.Also use standard method amplification and purifying PCR fragment (to comprise the attB site; Extremely stop from initial).Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, " clone crosses the threshold ".Plasmid pDONR201 conduct The part of technology is available from Invitrogen.

Embodiment 34a: the structure of carrier

The clone that will cross the threshold subsequently is used from the LR reaction with the purpose carrier one that comprises prolamine that is used for the rice conversion.This carrier form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and be intended to be used for carry out the Gateway box of recombinating the LR body in the aim sequence that has been cloned into the clone that crosses the threshold.The paddy prolamine promotor (SEQ ID NO:258) that is used for seed-specific expression is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, it is LBA4044 that the gained expression vector shown in Figure 27 is transformed into the Agrobacterium strain, and then is transformed into rice plant.Make rice plant's growth of conversion, the parameter that just describes below is then checked it.About the conversions of other farm crop referring to embodiment 40.

Embodiment 35: assessment and result

Produce about 15 to 20 T0 rice transformant independently.With former generation transformant be transferred to from tissue culture room and grow the greenhouse and gather in the crops the T1 seed.Reservation is with regard to genetically modified existence/the T1 offspring does not present isolating 7 incidents of 3:1 with regard to not existing.For each incident in these incidents,, select about 10 strains to comprise the T1 seedling of transgenosis (heterozygote and homozygote) and the T1 seedling that about 10 strains lack transgenosis (invalid zygote) by the expression of monitoring witness marking.According to further assessing about 4 T1 incidents for the appraisal procedure of identical (but the assessment of every incident is more individual) with T1.

To grow until heading stage under normal operation from the plant of 5 incidents.Use the hygroreceptor continuous monitoring soil humidity in the flowerpot that inserts several non-transgenic control plants of selecting at random.In first period, make flowerpot be saturated to 60% maximum value, thereby reduce the otherness of basin and basin.After flowerpot is saturated, stop to irrigate until obtaining to be lower than 20% soil moisture content.Then plant is watered again and reach 60% highest level until soil humidity once more.Then plant is taken a picture to assess following relevant and seed correlation parameter.

The root correlation parameter

Plant-growth is had in the flowerpot of clear bottom with permission observation root specially designed.In growing process, pass through the bottom digital camera document image of flowerpot.Use suitable software to derive the feature of root, for example total projection area (it can be relevant with total root volume), mean diameter and be higher than the length (the slightly length of root or the length of radicula) of the root of certain rugosity threshold value from the image that produces.

The measurement of seed correlation parameter

Gather in the crops sophisticated main panicle, counting, packed, use bar code label, under 37 ℃ in baking oven dry 3 days then.To the panicle threshing, collect all seeds then, count then.Use gas blower that full husk is separated with empty husk.Discard the sky husk, then the residue fraction is counted once more.On analytical balance, take by weighing the weight of full husk.Determine the number of full seed by the number of counting the full husk that behind separating step, stays.Measure total seed production by taking by weighing from the weight of all full husks of plant results.Measure total number seeds of every strain plant by counting from the number of the husk of plant results.Number and its gross weight according to the full seed of counting are calculated thousand seed weight (TKW).Harvest index among the present invention (HI) is defined as seed ultimate production and ground area (mm ²) between ratio multiply by the factor 10 ⁶The sum of every paniculiform flower is the ratio between seed sum and the paniculiform number of sophisticated master among the present invention.The full rate of the seed that defines among the present invention is the ratio (representing with per-cent) that the number of full seed accounts for seed (or Xiao Hua) sum.

Statistical analysis: F check

Below Table X be presented at GOS2 promotor control express down encoding D OF transcription factor nucleic acid transgenic plant the T2 assessment result and under the control of prolamine promotor, express the T2 assessment result of transgenic plant of the nucleic acid of encoding D OP transcription factor.Although do not show, on the T1 plant, obtained suitable result.Shown the p value that F checks at listed parameter in the table, and transgenic plant are with respect to the percentage difference of invalid zygote.

The result of Table X: T2 assessment

Except above-mentioned seed correlation parameter, compare with invalid zygote, following parameter also increases in transgenic plant: the mean diameter of total root biomass increase by 14%, radicula number (internal threshold) increase by 7%, thick radical order (internal threshold) increase by 36% and root increases by 8%.

Under the condition of slight drought stress, obtained The above results; Be expected under the normal or non-stress conditions and will obtain similar result.

Embodiment G:CKI

Embodiment 36: the clone of the rice gene of coding CKI4 polypeptide

According to manufacturers instruction, use HybriZAP-2.1 test kit (Stratagene, the rice cell suspension culture cDNA library of cloning in pAD-Gal4-2.1 carrier La Jolla, California USA) is as template, by the rice gene of pcr amplification coding CKI4 polypeptide.The average inset size in this library is 1.5kb, and clone's original number is about 2 x 10 ⁶Pfu.10 ¹⁰After the amplification first time of pfu/ml, initial titer is defined as 4 x 10 ⁶Pfu/ml.After plasmid extracts, the 200ng template is used for 50 μ l PCR mixtures.Primer (the SEQ ID NO:284 that will comprise the AttB site that is used for the Gateway reorganization; 5 '-GGGGGACAAGTTTGTACAAAAAAGCAGGCTTCACAATGGGCAAGTACATGCGCAAG GCC-3 ') and (SEQ ID NO:285 justice is arranged, and initiator codon indicates with runic, and the AttB1 site indicates with italic:; Oppositely, complementation; The AttB2 site indicates with italic: 5 '-GGGGACCCACTTTGTACAAGAAAGCTGGG TGGAGCAGAGAGGTCCATGGTGCCC-3 ') be used for pcr amplification.Under standard conditions, use Hifi TaqDNA polysaccharase to carry out PCR.Also use the PCR fragment of standard method amplification and purifying 662bp (to comprise the attB site; From initial to stopping 585bp).Carry out the first step BP reaction of Gateway method then, during this step, PCR fragment and pDONR201 plasmid carry out reorganization in the body, thereby produce, according to the Gateway nomenclature, " clone crosses the threshold ".Plasmid pDONR201 is as Gateway

The part of technology is available from Invitrogen.

Embodiment 37: the structure of carrier

The clone that will cross the threshold subsequently is used from the LR reaction with the purpose carrier one that is used for the rice conversion.This carrier form with functional element in the T-DNA border comprises: plant selectable marker, the marker expression box that can screen and two Gateway boxes with opposed orientation that are intended to be used for carry out reorganization in the LR body with the aim sequence that is cloned into the clone that crosses the threshold.These two Gateway boxes are separated by noncoding DNA (being the 315bp fragment of tobacco matrix attachment regions (MAR) under this situation, NCBI referenceU67919, the fragment from 774 to 1088bp), thereby are transcribing this mRNA formation hairpin structure of back promotion.The rice RP6 prolamine promotor (SEQ IDNO:281) that is used for endosperm specificity expression is positioned at the upstream of a Gateway box, with this Gateway box opposed orientation.

The clone that also will cross the threshold is used from the LR reaction with another purpose carrier one that is used for the rice conversion.Except the rice β expansion protein promoter with SEQ ID NO:282 replaces the RP6 prolamine promotor, this carrier is identical with above-described carrier.

After the LR reconstitution steps, it is LBA4044 that the expression vector (about two carriers, referring to Figure 32) of two gained is transformed into the Agrobacterium strain, and then is transformed into rice plant.Make rice plant's growth of conversion, with regard to the parameter of describing in

embodiment

38 and 39 it is checked then.About the conversions of other farm crop referring to embodiment 40.

Embodiment 38: the description of phenotype appraisal procedure

Produce about 15 to 20 T0 rice transformant independently.With former generation transformant be transferred to the greenhouse to grow and to gather in the crops the T1 seed from tissue culture room.Keep the T1 offspring with regard to genetically modified existence/present with regard to not existing isolating 4 to 5 incidents of 3:1.For each incident in these incidents,, select about 10 strains to comprise the T1 seedling of transgenosis (heterozygote and homozygote) and the T1 seedling that about 10 strains lack transgenosis (invalid zygote) by the expression of monitoring witness marking.Transgenic plant and suitable control plant are grown at random position side by side.From sowing time to the ripening stage, plant is passed through the digital imagery chamber for several times.On each time point, to every strain plant from least 6 different angle shot digital pictures (2048x1536 pixel, 1,000 6 hundred ten thousand pigments).

According to further being evaluated at the similar events as in T1 assessed at T2 in generation for identical appraisal procedure with T1.

The measurement of seed correlation parameter

Results, counting, packed, with the sophisticated main panicle of bar code label, under 37 ℃ in baking oven dry 3 days then.To the panicle threshing, collect all seeds then, count then.Use gas blower that full husk is separated with empty husk.Discard the sky husk, then the residue fraction is counted once more.On analytical balance, take by weighing the weight of full husk.Determine the number of full seed by the number of counting the full husk that behind separating step, stays.Measure total seed production by taking by weighing from the weight of all full husks of plant results.Measure total number seeds of every strain plant by counting from the number of the husk of plant results.Harvest index among the present invention (HI) is defined as seed ultimate production and ground area (mm ²) between ratio multiply by the factor 10 ⁶The sum of every paniculiform flower is the sum of seed and the ratio between the paniculiform number of sophisticated master in the present invention.The full rate of the seed that defines among the present invention is the ratio (representing with per-cent) that the number of full seed accounts for seed (or Xiao Hua) sum.

Statistical analysis: F check

Embodiment 39: the assessment result that has the transgenosis rice plant that the CKI4 of minimizing expresses in endosperm

Among the table Y below the assessment of the measurement result (number of seed production, full seed, seed sum and every paniculiform number of spending) that has the transgenic plant that the CKI4 of minimizing expresses in endosperm is shown in.The number, average percent that has shown the plant that has increase on parameter increases and the P value in T2 generation, and with have the result who obtains in the transgenic plant of CKI4 expression of minimizing and compare by using β to expand protein promoter (preferentially in root tissue, expressing).

The result shows that the CKI4 expression that reduces in the endosperm causes, express the transgenic plant of (using β expansion protein promoter) with invalid zygote and the CKI4 that preferentially in root tissue, has a minimizing and compare, have seed weight, full seed number, seed sum and every paniculiform several plant of spending of remarkable increase.

Table Y: the assessment of the measurement result that in endosperm, has the transgenic plant that the CKI4 of minimizing expresses

Embodiment 40: the conversion of corn, wheat, soybean, rape (Rapseed) and alfalfa

The conversion of corn

Use is carried out the conversion of corn (Zea mays) by the improving one's methods of method that people such as Ishida (1996) Nature Biotech 14 (6): 745-50 describes.Transforming in corn is that genotype is dependent, has only the special genes type to be easy to transform and regenerate.Inbred lines A188 (University ofMinnesota) or be the good source of the donor material that is used to transform with A188 as parent's hybrid, but also can successfully use other genotype.In (DAP) after the pollination about 11 days length when immature embryo is about 1 to 1.2mm the time, from maize plant results tassel.Immature embryo and the agrobacterium tumefaciens (Agrobacterium tumefaciens) that comprises expression vector are cultivated altogether, transgenic plant are taken place to recover by organ.The embryo culture that cuts off on callus inducing medium, is cultivated on the corn regeneration culture medium that comprises selective agent (for example imidazolone, but can use different selective markers) then.Under 25 ℃, hatching 2 to 3 weeks of culture dish under the situation of illumination, or producing until bud.The bud of green is transferred to the maize rooting substratum from each embryo, under 25 ℃, hatches 2-3 week, produce until root.The bud that to take root migrates in the greenhouse soil then.From showing the resistance of selective agent and the plant that comprises single copy T-DNA inset are produced the T1 seed.

Wheat transforms

Use is carried out the conversion of wheat by the method that people such as Ishida (1996) Nature Biotech 14 (6): 745-50 describes.Usually Cultivar Bobwhite (can be from CIMMYT, Mexico obtains) is used for transforming.Immature embryo and the agrobacterium tumefaciens that comprises expression vector are cultivated altogether, transgenic plant are taken place to recover by organ.After hatching with Agrobacterium, embryo is carried out vitro culture on callus inducing medium, on the regeneration culture medium that comprises selective agent (for example imidazolone, but can use different selective markers), cultivate then.Under 25 ℃, hatching 2 to 3 weeks of culture dish under the situation of illumination, or producing until bud.The bud of green is transferred to root media from each embryo, under 25 ℃, hatches 2-3 week, produce until root.The bud that to take root migrates in the greenhouse soil.From showing the resistance of selective agent and the plant that comprises single copy T-DNA inset are produced the T1 seed.

Soybean transforms

According to Texas A﹠amp; The soybean transformation of improving one's methods of the method for describing among the M patent US 5,164,310.Several commercialization soybean varieties are easy to transform by this method.Usually Cultivar Jack (can obtain from Illinois Seed foundation) is used for transforming.Soybean seeds is sterilized so that external sowing.Cut hypocotyl, radicle and a slice cotyledon from 7 age in days seedling.Allow epicotyl and remaining cotyledon further growth produce armpit joint (axillary node).Cut these armpits joint, with it with the agrobacterium tumefaciens incubation that comprises expression vector.After cultivating processing altogether, the washing explant is transferred to the selection substratum then.Cut the regenerated bud, be placed on the bud elongation medium.The bud that length is no more than 1cm places on the root media and produces until root.The bud that to take root is transferred in the soil in greenhouse.From showing the resistance of selective agent and the plant that comprises single copy T-DNA inset are produced the T1 seed.

The conversion of rape/Canola

Cotyledon petiole of the seedling of 5-6 age in days and hypocotyl transform it according to people such as Babic (1998, Plant Cell Rep 17:183-188) as the explant of tissue culture.Commercial Cultivar Westar (Agriculture Canada) is the standard variety that is used to transform, but also can use other kinds.The Canola seed is carried out surface sterilization to carry out external sowing.Cut cotyledon petiole explant from external seedling, then by terminal the immersion in the bacterial suspension of the otch of cotyledon petiole explant inoculated Agrobacterium (comprising expression vector) with the cotyledon that adheres to.Then with explant under illumination in 23 ℃, 16 hours, on the MSBAP-3 substratum that comprises 3mg/l BAP, 3% sucrose, 0.7% Phytagar, cultivated 2 days.After cultivating 2 days altogether with Agrobacterium, the cotyledon petiole explant is transferred on the MSBAP-3 substratum that comprises 3mg/l BAP, cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid (300mg/l) and cultivated 7 days, cultivate until shoot regeneration having on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent then.When the length of bud is 5-10mm, it is cut off, be transferred to bud elongation medium (MSBAP-0.5 comprises 0.5mg/l BAP) then.The bud of the about 2cm of length is transferred to root media (MS0) to carry out root induction.The bud that to take root migrates in the soil in greenhouse.From showing the resistance of selective agent and the plant that comprises single copy T-DNA inset are produced the T1 seed.

The conversion of alfalfa

Use the method for people such as (, 1999 Plant Physiol 119:839-847) McKersie to transform the regeneration clone of alfalfa (Medicago sativa).Regeneration of alfalfa and conversion are that genotype is dependent, thereby need the regenerated plant.The method that is used to obtain aftergrowth has been described.For example, these aftergrowths any other commercial alfalfa kind that can be selected from Cultivar Rangelander (Agriculture Canada) or describe by Brown DCW and A Atanassov (1985.Plant Cell Tissue Organ Culture 4:111-112).Selectively, selected RA3 kind (University of Wisconsin) to be used for tissue culture (people such as Walker, 1978 Am J Bot65:654-659).Cotyledon petiole explant and agrobacterium tumefaciens C58C1 pMP90 people such as (, 1999 Plant Physiol 119:839-847) McKersie or the overnight culture of LBA4404 that comprise expression vector are cultivated altogether.Explant is being comprised 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K in the dark ₂SO ₄With cultivated altogether 3 days on the SH inducing culture of 100 μ m Syringylethanones.At the Murashige-Skoog of half strength substratum (Murashige and Skoog, 1962) washing explant in, then with its kind do not contain Syringylethanone but have suitable selective agent and the identical SH inducing culture of suitable microbiotic (with the inhibition growth of Agrobacterium) on.After several weeks, somatic embryo is transferred to the BOi2Y that does not contain growth regulator, antibiotic-free but contain 50g/L sucrose grows in the substratum.On the Murashige-Skoog of half strength substratum, sprout somatic embryo subsequently.The sprigging that to take root is gone into flowerpot and is grown in the greenhouse.From showing the resistance of selective agent and the plant that comprises single copy T-DNA inset are produced the T1 seed.

Claims

1. be selected from following isolating SYR albumen:

(a) polypeptide of expression among the SEQ ID NO 44,

(b) has the polypeptide of aminoacid sequence, described aminoacid sequence by the preferred sequence that increases progressively have with SEQ ID NO 44 in the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher sequence identity of expression

(c) (a) or (b) in the derivative of polypeptide of definition.

2. isolated nucleic acid sequences, it comprises:

(a) nucleotide sequence of representing by SEQ ID NO:43, or its complementary strand;

(b) nucleotide sequence of the aminoacid sequence represented by SEQ ID NO:44 of coding;

(c) can (preferably under stringent condition) with (a) or the nucleotide sequence of nucleic acid array hybridizing (b), this hybridization sequences optimized encoding SYR albumen;

(d) nucleic acid of the allele variant of conduct (a) or nucleotide sequence (b);

(e) nucleic acid of the alternative splicing variant of conduct (a) or nucleotide sequence (b);

(f) have with (a) or (b) in the sequence 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% of definition or the nucleotide sequence of 99% sequence identity.

3. be used for comparing seed production that increases plant and/or the method that increases the growth velocity of plant with corresponding wild type plant, this method is included in the plant of regulating the expression of nucleic acids of coding SYR polypeptide or its homologue in the plant and randomly selecting to have the growth characteristics of improvement; Condition is that described SYR albumen or its homologue are not the albumen of SEQ ID NO:26.

4. the method for claim 3 is wherein passed through, and preferably in the locus of the gene of coding SYR polypeptide or its homologue, imports the adjusting that genetic modification is realized described expression.

5. the method for claim 4, wherein pass through: a method in T-DNA activation, TILLING, directed mutagenesis, homologous recombination or the orthogenesis realizes described genetic modification.

6. be used for comparing the method that increases seed production and/or increase growth velocity with corresponding wild type plant, this method is included in and imports and express SYR nucleic acid or its variant in the plant; Condition is that described SYR albumen or its homologue are not the albumen of SEQ ID NO:26.

7. the method for claim 6, the proteic homologue of SYR of wherein said nucleic acid encoding SEQ ID NO:2, proteic lineal homologue of SYR or the collateral line homologue of preferred described nucleic acid encoding SEQ ID NO:2.

8. the method for claim 6, wherein said variant be SYR nucleic acid a part or can with the sequence of SYR nucleic acid hybridization, described part or hybridization sequences encode about 65 to about 200 amino acid whose polypeptide, described polypeptide comprises and is rich in leucic structural domain, before the described structural domain conservative tripeptides primitive 1 (among the SEQ ID NO:6,7,8 or 9 one)), and follow conservative primitive 2 (SEQ ID NO:10) afterwards, and preferably also follow conservative primitive 3 (SEQ IDNO:11).

9. the method for each of claim 6 to 8, wherein said nucleic acid comprises the conservative primitive of SEQ ID NO:6, SEQ ID NO:10 and SEQ ID NO:11, wherein the primitive of SEQ ID NO:10 is VLAFMPT, wherein the primitive of SEQ ID NO:11 is PYL, and preferred wherein said nucleic acid comprises the sequence of SEQ ID NO:1.

10. the method for each of claim 6 to 9, wherein said SYR nucleic acid or its variant are crossed in plant and are expressed.

11. the method for each of claim 6 to 10, wherein said SYR nucleic acid or its variant are plant origins, and preferably from monocotyledons, more preferably from Gramineae, most preferably this nucleic acid is from rice.

12. the method for each of claim 6 to 11, wherein said SYR nucleic acid or its variant effectively are connected with constitutive promoter.

13. the method for claim 12, wherein said constitutive promoter are GOS2 promotor or high mobility group protein promotor.

14. the method for each of claim 1 to 13, the seed production of wherein said increase is selected from: the harvest index of the seed gross weight of increase, the full seed number of increase, the full rate of seed or increase.

15. the method for each of claim 1 to 14, the growth velocity of wherein said increase are included in the seed production of the increase that obtains under the situation that flowering time do not postpone at least.

16. the method for each of claim 1 to 15, the wherein described plant of growth under non-stress conditions.

17. the method for each of claim 1 to 15, the wherein described plant of growth under the abiotic stress condition.

18. the method for claim 17, wherein said abiotic stress condition is the osmotic stress condition.

19. the plant or the vegetable cell that can obtain by each method of claim 1 to 18.

20. a construct, it comprises:

(i) SYR nucleic acid or its variant;

(iii) transcription termination sequence,

Condition is the do not encode albumen of SEQ ID NO:26 of described SYR nucleic acid.

21. the construct of claim 20, wherein said control sequence is a constitutive promoter.

22. the construct of claim 21, wherein said constitutive promoter are GOS2 promotor or high mobility group protein (HMGP) promotor.

23. the construct of claim 22, wherein said GOS2 promotor is the promotor of being represented by SEQ IDNO:5.

24. the construct of claim 22, wherein said HMGP promotor is the promotor of being represented by SEQ IDNO:33.

25. the construct plant transformed or the vegetable cell of each of use claim 20 to 24.

26. be used to produce the method for comparing the transgenic plant of the output with increase with corresponding wild type plant, this method comprises:

(a) in plant or vegetable cell, import and express SYR nucleic acid or its variant; With

(b) culturing plants cell under the condition that promotes plant-growth and growth,

Condition is the do not encode albumen of SEQ ID NO:26 of described SYR nucleic acid or its variant.

27. transgenic plant or vegetable cell, it has the seed production of the increase that is caused by the SYR nucleic acid or its variant that import in the described plant and/or the growth velocity of increase, and condition is the do not encode albumen of SEQ ID NO:26 of described SYR nucleic acid or its variant.

28. claim 19,25 or 27 transgenic plant or vegetable cell, wherein said plant is a monocotyledons, for example sugarcane or wherein this plant be cereal, for example rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum, or wherein said transgenic plant cells derives from monocotyledons, for example sugarcane or wherein this plant be for example rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum of cereal.

29. claim 19,25,27 or 28 each the part gathered in the crops of plant.

30. the part gathered in the crops of the plant of claim 29, wherein said gather in the crops the part be seed.

31. be directed to the plant of claim 27 and/or derive from the product of the part gathered in the crops of the plant of claim 29 or 30.

32.SYR nucleic acid/gene or its variant or SYR polypeptide or its homologue are used for comparing with corresponding wild type plant the purposes of raising seed production.

33. the purposes of claim 32, wherein said seed production is: in the harvest index of the seed gross weight of increase, the full seed number of increase or increase one or multinomial.

34.SYR nucleic acid/gene or its variant or SYR polypeptide or its homologue are used for comparing with corresponding wild type plant and improve the purposes of plant to the resistance of abiotic stress.

35.SYR nucleic acid/gene or its variant or SYR polypeptide or its homologue are as the purposes of molecule marker.。

36. be used for comparing with control plant the method for the growth characteristics of improvement plant, this method is included in the plant that increases the expression of nucleic acids of coding FG-GAP polypeptide or its homologue in the plant and randomly select to have the growth characteristics of improvement.

37. the method for claim 36 is wherein passed through, and preferably in the locus of the gene of coding FG-GAP polypeptide or its homologue, imports the expression that genetic modification is realized described increase.

38. the method for claim 37 is wherein passed through: a method in T-DNA activation, TILLING, directed mutagenesis, homologous recombination or the orthogenesis realizes described genetic modification.

39. be used for comparing with corresponding wild type plant the method for improvement growth characteristics, this method is included in and imports and express FG-GAP nucleic acid or its variant in the plant.

40. the method for claim 39, the proteic homologue of FG-GAP of wherein said nucleic acid encoding SEQ ID NO:46, proteic lineal homologue of FG-GAP or the collateral line homologue of preferred described nucleic acid encoding SEQ ID NO:46.

41. the method for claim 39, wherein said variant be FG-GAP nucleic acid a part or can with the sequence of FG-GAP nucleic acid hybridization, described part or hybridization sequences coded polypeptide, described polypeptide comprise signal peptide, one or more FG-GAP structural domain and are arranged in the membrane spaning domain of proteinic C end half part.

42. the method for each of claim 39 to 41, wherein said nucleic acid comprise one or more in the conservative primitive of SEQIDNO:50 to 52.

43. the method for each of claim 39 to 42, wherein said FG-GAP nucleic acid or its variant are crossed in plant and are expressed.

44. the method for each of claim 39 to 43, wherein said FG-GAP nucleic acid or its variant are plant origins, and preferably from dicotyledons, more preferably from Cruciferae, most preferably this nucleic acid is from Arabidopis thaliana.

45. the method for each of claim 39 to 44, wherein said FG-GAP nucleic acid or its variant effectively are connected with constitutive promoter.

46. the method for claim 45, wherein said constitutive promoter are the GOS2 promotors.

47. the method for each of claim 36 to 46, the growth characteristics of wherein said improvement are the output that increases.

48. the method for claim 47, the output of wherein said increase are the seed productions that increases.

49. the method for claim 48, the seed production of wherein said increase is selected from: the seed gross weight of increase, the full seed number of increase or the harvest index of increase.

50. plant, plant part or the vegetable cell that can obtain by each method of claim 36 to 49.

51. a construct, it comprises:

(a) FG-GAP nucleic acid or its variant;

(b) can drive one or more control sequences that the nucleotide sequence of (a) is expressed; Randomly

(c) transcription termination sequence,

Condition is that described construct is not a pPZP type gene construct.

52. the construct of claim 51, wherein said control sequence is a constitutive promoter.

53. the construct of claim 52, wherein said constitutive promoter are the GOS2 promotors.

54. sharp 53 the construct that requires, wherein said GOS2 promotor is the promotor by 1 to 2193 expression of the Nucleotide among the SEQ ID NO:49.

55. construct plant transformed, plant part or the vegetable cell of each of use claim 51 to 54.

56. be used to produce the method for comparing the transgenic plant of the output with increase with corresponding wild type plant, this method comprises:

(a) in plant or vegetable cell, import and express FG-GAP nucleic acid or its variant; With

(b) culturing plants cell under the condition that promotes plant-growth and growth.

57. transgenic plant, plant part or vegetable cell, it has the growth characteristics of the improvement that is caused by the FG-GAP nucleic acid or its variant that import in the described plant.

58. claim 50,55 or 57 transgenic plant, plant part or vegetable cell, wherein said plant is a monocotyledons, for example sugarcane or wherein this plant be cereal, for example rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum, or wherein said transgenic plant cells derives from monocotyledons, for example sugarcane or wherein this plant be cereal, for example rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum.

59. claim 50,55,57 or 58 each the part gathered in the crops of plant.

60. the part gathered in the crops of the plant of claim 59, wherein said gather in the crops the part be seed.

61. derive from the plant of claim 58 and/or derive from the product of the part gathered in the crops of the plant of claim 59 or 60.

62.FG-GAP nucleic acid/gene or its variant or FG-GAP polypeptide or its homologue are used for comparing with corresponding wild type plant the growth characteristics of improvement plant, preferably improve the purposes of output, particularly seed production.

63. the construct of each of claim 51 to 54 is used for comparing with corresponding wild type plant the growth characteristics of improvement plant, preferably improves the purposes of output, particularly seed production.

64. the purposes of claim 62 or 63, wherein said seed production is: in the harvest index of the seed gross weight of increase, the full seed number of increase or increase one or multinomial.

65.FG-GAP nucleic acid/gene or its variant or FG-GAP polypeptide or its homologue are as the purposes of molecule marker.

66. an isolating FG-GAP albumen, it is selected from:

(a) by the albumen of the nucleic acid encoding of SEQ ID NO:72;

(b) albumen that comprises signal sequence, one or more FG-GAP structural domain and be arranged in the membrane spaning domain of proteinic C end half part, wherein said albumen comprises at least one among SEQ ID NO:73 to the SEQ ID NO:76;

(c) (a) or (b) in the active fragments of aminoacid sequence of definition, the membrane spaning domain that this active fragments comprises signal sequence, one or more FG-GAP structural domain and is arranged in proteinic C end half part.

67. the proteic nucleic acid of separated coding FG-GAP, it is selected from:

(i) nucleic acid of representing by SEQ ID NO:72;

Proteic nucleic acid of definition in (a) to (c) above (ii) encoding;

(iii) can with the nucleotide sequence of top (i) or nucleic acid array hybridizing (ii) (preferably under stringent condition), this hybridization sequences optimized encoding albumen, described albumen comprise signal peptide, one or more FG-GAP structural domain and are arranged in the membrane spaning domain of this proteinic C end half part;

(iv) as (i) nucleic acid to the allele variant of (iii) nucleotide sequence;

(vi) Shang Mian (i) is to (part of the nucleotide sequence of any one v), this part optimized encoding protein, described protein comprise signal peptide, one or more FG-GAP structural domain and be arranged in the membrane spaning domain of this proteinic C end half part.

68. isolating CYP90B albumen, it is selected from:

(i) by the albumen of the nucleic acid encoding of SEQ ID NO:117;

(ii) comprise: (i) CYP structural domain A to D; (ii) N holds hydrophobic anchor structure territory; (iii) translocation domain; (iv) in the A structural domain, allow to produce the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes in any position; And has albumen with the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ ID NO:118,99% identity by the preferred sequence that increases progressively.

69. the proteic nucleic acid of separated coding CYP90B, it is selected from:

(i) nucleic acid represented of SEQ ID NO:117;

(i) of the claim 68 of (ii) encoding and the (ii) middle proteinic nucleic acid that defines;

(iii) has the nucleic acid at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% represented with SEQ ID NO:117,99% or the nucleic acid of higher identity by the preferred sequence that increases progressively;

(iv) can be under stringent condition and top (i) nucleotide sequence to (iii) nucleic acid array hybridizing, this hybridization sequences proteins encoded, described albumen comprise (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow to produce the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes in any position; And have and the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ ID NO:118,99% or higher identity by the preferred sequence that increases progressively;

((i) is to (part of each nucleotide sequence v), this part proteins encoded, described albumen comprise (i) CYP structural domain A to D vi); (ii) N holds hydrophobic anchor structure territory; (iii) translocation domain; (iv) in the A structural domain, allow to produce the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes in any position; And have and the aminoacid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ ID NO:118,99% identity by the preferred sequence that increases progressively.

70. be used for comparing the method that increases plant biomass with suitable control plant, described method is included in the non-constitutive expression that increases the nucleic acid of Codocyte cytochrome p 450 (CYP) monooxygenase CYP90B or its homologue in the plant, randomly selection has the plant of the output of increase, and wherein said CYP90B polypeptide or homologue comprise: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes to take place in any position.

71. the method for claim 70, wherein said CYP90B polypeptide or its homologue also comprise: (i) have the sequence greater than 50% identity with SEQ ID NO:78; (ii) steroid 22 α hydroxylase enzymatic activities.

72. the method for claim 70 or 71 is wherein passed through, and preferably in the locus of the gene of coding CYP90B polypeptide or its homologue, imports the non-constitutive expression that genetic modification is realized described increase.

73. the method for claim 72 is wherein passed through: a method in T-DNA activation, TILLING, directed mutagenesis or the orthogenesis realizes described genetic modification.

74. be used for comparing the method that increases plant biomass with suitable control plant, this method is included in and imports in the plant and non-composing type ground expression CYP90B nucleic acid or its variant.

75. the method for claim 74, wherein said variant are the parts of CYP90B nucleic acid, this part coded polypeptide, and described polypeptide comprises: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes to take place in any position.

76. the method for claim 74, wherein said variant be can with the sequence of CYP90B nucleic acid hybridization, this hybridization sequences coded polypeptide, described polypeptide comprises: (a) CYP structural domain A to D; (b) N holds hydrophobic anchor structure territory; (c) translocation domain; (d) in the A structural domain, allow the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser that an amino acid changes to take place in any position.

77. the method for each of claim 74 to 76, wherein said CYP90B nucleic acid or its variant are plant origins, preferably from monocotyledons, more preferably from Gramineae, more preferably from Oryza, most preferably from rice.

78. the method for each of claim 74 to 77, proteic lineal homologue of CYP90B or the collateral line homologue of wherein said variant coding SEQID NO:78.

79. the method for each of claim 74 to 78, wherein said CYP90B nucleic acid or its variant effectively are connected with non-constitutive promoter.。

80. the method for claim 79, wherein said non-constitutive promoter is a seed specific promoters, and preferred described seed specific promoters is an endosperm specificity promoter, and more preferably described endosperm specificity promoter is the prolamine promotor.

81. the method for claim 80, wherein said endosperm specificity promoter is a rice RP6 prolamine promotor, more preferably endosperm specificity promoter is by representing that to the similar basically nucleotide sequence of SEQ ID NO:109 most preferably endosperm specificity promoter is the promotor of being represented by SEQ ID NO:109.

82. the method for each of claim 70 to 81, the output of wherein said increase is selected from: in the harvest index (HI) of the seed ultimate production of increase or increase one or two, wherein each is all for suitable control plant.

83. the method for claim 79, wherein said non-constitutive promoter is a seed specific promoters, and preferred described seed specific promoters is embryo/aleuron specificity promoter, and more preferably described embryo/aleuron specificity promoter is the oleosin promotor.

84. the method for claim 83, wherein said embryo/aleuron specificity promoter is a rice oleosin 18kDa promotor, more preferably embryo/aleuron specificity promoter is by representing that to the similar basically nucleotide sequence of SEQ ID NO:110 most preferably embryo/aleuron specificity promoter is the promotor of being represented by SEQ IDNO:110.

85. claim 70 to 79,83 or 84 each method, the output of wherein said increase is selected from: in the seed length of the thousand seed weight of increase (TKW), the seed area that increases or increase one or multinomial, wherein each is all for the control plant that suits.

86. the method for each of claim 70 to 85, wherein said plant is a monocotyledons.

87. plant, plant part or the vegetable cell that can obtain by each method of claim 70 to 86.

88. the purposes of construct in each method of claim 74 to 86, described construct comprises:

(i) CYP90B nucleic acid or its variant as defined above;

The nucleotide sequence that (ii) can drive (i) carries out one or more control sequences of non-constitutive expression; Randomly

(iii) transcription termination sequence.

89. the purposes of claim 88, wherein said control sequence is a non-constitutive promoter.

90. the purposes of claim 89, wherein said non-constitutive promoter is a seed specific promoters, and preferred described seed specific promoters is an endosperm specificity promoter, and more preferably described endosperm specificity promoter is the prolamine promotor.

91. the purposes of claim 90, wherein said endosperm specificity promoter is a rice RP6 prolamine promotor, more preferably endosperm specificity promoter is by representing that to the similar basically nucleotide sequence of SEQ ID NO:109 most preferably endosperm specificity promoter is the promotor of being represented by SEQ ID NO:109.

92. with being included in the CYP90B nucleic acid under the RP6 prolamine promotor control or construct plant transformed, plant part or the vegetable cell of its variant.

93. the purposes of claim 89, wherein said non-constitutive promoter is a seed specific promoters, and preferred described seed specific promoters is embryo/aleuron specificity promoter, and more preferably described embryo/aleuron specificity promoter is the oleosin promotor.

94. the purposes of claim 93, wherein said embryo/aleuron specificity promoter is a rice oleosin 18kDa promotor, more preferably embryo/aleuron specificity promoter is by representing that to the similar basically nucleotide sequence of SEQ ID NO:110 most preferably embryo/aleuron specificity promoter is the promotor of being represented by SEQ IDNO:110.

95. with being included in the CYP90B nucleic acid under the oleosin 18kDa promotor control or construct plant transformed, plant part or the vegetable cell of its variant.

96. be used to produce the method for comparing the transgenic plant of the output with increase with suitable control plant, this method comprises:

(a) CYP90B nucleic acid or its variant are expressed in importing and non-composing type ground in plant or vegetable cell; With

97. compare the transgenic plant of the output with increase with suitable control plant, the output of described increase is by the described plant of importing and CYP90B nucleic acid or its variant of non-constitutive expression cause therein.

98. claim 87,92,95 or 97 each transgenic plant, wherein said plant is a monocotyledons, for example sugarcane or wherein this plant be cereal, for example rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum.

99. claim 87,92,95,97 or 98 each the part gathered in the crops of plant.

100. the part gathered in the crops of the plant of claim 99, wherein said gather in the crops the part be seed.

Derive from the plant of claim 98 and/or derive from the product of the part gathered in the crops of the plant of claim 99 or 100.

Claim 90 or 91 construct are used for comparing the purposes that increases plant biomass with suitable control plant.

The purposes of claim 102, the output of wherein said increase is selected from: among the seed ultimate production of increase or the HI of increase one or two, wherein each is all for suitable control plant.

Claim 93 or 94 construct are used for comparing the purposes that increases plant biomass with suitable control plant.

The purposes of claim 104, the output of wherein said increase is selected from: in the average seed area of the TKW of increase, increase or the average seed length of increase one or multinomial, wherein each is all for suitable control plant.

Be used for comparing the method for the number seeds that increases plant with suitable control plant, this method comprises the expression of nucleic acids that preferentially increases coding CDC27 polypeptide in the stem end meristematic tissue of plant, randomly selection has the plant of the number seeds of increase, and wherein said CDC27 polypeptide is at the NH of polypeptide ₂The TPR structural domain that has at least one inactivation in the end regions.

The method of claim 106 is wherein passed through, and preferably is being coded in the NH of polypeptide ₂Have in the end regions in the locus of gene of CDC27 polypeptide of TPR structural domain of at least one inactivation, import the expression that genetic modification is realized described increase.

The method of claim 107 wherein realizes described genetic modification by directed mutagenesis or orthogenesis.

Be used for comparing with suitable control plant the method for the number seeds that increases plant, this method is included in the nucleic acid that imports and preferentially express therein coding CDC27 polypeptide in the stem end meristematic tissue of plant, and wherein said CDC27 polypeptide is at the NH of polypeptide ₂The TPR structural domain that has at least one inactivation in the end regions.

110. the method for claim 109, the nucleic acid of wherein said importing are the splice variant or the allele variant of the nucleic acid represented by SEQ ID NO:129 or SEQ ID NO:131, described splice variant or allele variant are coded in the NH of polypeptide ₂The CDC27 polypeptide that has the TPR structural domain of at least one inactivation in the end regions.

111. the method for claim 109 or 110, the nucleic acid of wherein said importing can be hybridized with the splice variant or the allele variant of nucleic acid of being represented by SEQ ID NO:129 or SEQ ID NO:131 or claim 110, and wherein said hybridization sequences is coded in the NH of polypeptide ₂The CDC27 polypeptide that has the TPR structural domain of at least one inactivation in the end regions.

112. the method for each of claim 106 to 111, the wherein said NH that is coded in polypeptide ₂CDC27 nucleic acid or this polypeptide itself of CDC27 polypeptide that has the TPR structural domain of at least one inactivation in the end regions is plant origin, preferably from dicotyledons, preferably from the plant of Cruciferae, more preferably from Arabidopis thaliana.

113. the method for each of claim 106 to 112, the wherein said NH that is coded in polypeptide ₂The lineal homologue or the collateral line homologue that have the CDC27 polypeptide that the CDC27 nucleic acid encoding of CDC27 polypeptide of the TPR structural domain of at least one inactivation represented by SEQ ID NO:130 or SEQ ID NO:132 in the end regions.

114. the method for each of claim 109 to 113, the wherein said NH that is coded in polypeptide ₂The CDC27 nucleic acid and the stem end meristematic tissue promotor of CDC27 polypeptide that has the TPR structural domain of at least one inactivation in the end regions, preferred early stage stem end meristematic tissue promotor effectively connects.

115. the method for claim 114, wherein said stem end meristematic tissue promotor is the OSH1 promotor.

116. plant, plant part or the vegetable cell that can obtain by each method of claim 106 to 115.

117. the purposes of construct in each method of claim 109 to 115, described construct comprises:

(i) be coded in the NH of polypeptide ₂The CDC27 nucleic acid of CDC27 polypeptide that has the TPR structural domain of at least one inactivation in the end regions; With

The one or more control sequences of expression that (ii) can be preferentially in the stem end meristematic tissue of plant, increase the nucleotide sequence of (i); Randomly

(iii) transcription termination sequence.

118. the purposes of the construct of claim 117, wherein said control sequence are the OSH1 promotors.

119. construct plant transformed, plant part or vegetable cell with claim 117 or 118.

120. be used to produce the method for comparing the transgenic plant of the number seeds with increase with suitable control plant, this method comprises:

(a) importing and the effective NH that be connected, that be coded in polypeptide of expression and stem end meristematic tissue specificity promoter in plant ₂The CDC27 nucleic acid of CDC27 polypeptide that has the TPR structural domain of at least one inactivation in the end regions; With

121. compare the transgenic plant of the number seeds with increase with suitable control plant, the number seeds of described increase is that wherein said CDC27 polypeptide is at the NH of polypeptide because the CDC27 nucleic acid of coding CDC27 polypeptide causes ₂The TPR structural domain that has at least one inactivation in the end regions, described CDC27 nucleic acid effectively is connected with stem end meristematic tissue specificity promoter.

122. claim 116,119 or 121 transgenic plant, wherein said plant is a monocotyledons, for example sugarcane or wherein this plant be cereal, for example rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum.

123. claim 116,119,121 or 122 each the part gathered in the crops of plant.

124. the part gathered in the crops of claim 123, wherein said gather in the crops the part be seed.

125. derive from the plant of claim 122 and/or derive from the product of the part gathered in the crops of the plant of claim 123 or 124.

126. the CDC27 nucleic acid of the coding CDC27 polypeptide that effectively is connected with stem end meristematic tissue specificity promoter or this polypeptide are used for comparing with suitable control plant the purposes of the number seeds that increases plant, wherein said CDC27 polypeptide is at the NH of polypeptide ₂The TPR structural domain that has at least one inactivation in the end regions.

127. be used for comparing the method that increases monocotyledonous seed production with the seed production of suitable control plant, this method comprises preferentially increases the expression of nucleic acids that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain in monocotyledonous endosperm tissue.

128. the method for claim 127, wherein said polypeptide also comprise a primitive in the following primitive: primitive 1:QGQ V/I GG; Or primitive 2:ILSLSGSFLPPPAPP; Or primitive 3:NATYERLP; Or primitive 4:SFTNVAYERLPL, have 0 or 1 amino acid and change in any position; Or primitive 5:GRFEILSLTGSFLPGPAPPGSTGLTIYLAGGQGQVVGGSVVG, have 0,1 or 2 amino acid and change in any position.

129. the method for claim 127 or 128 is wherein passed through, and preferably comprises in the locus of gene of polypeptide of AT hook structure territory and DUF296 structural domain at coding, imports the expression that genetic modification is realized described increase.

130. the method for claim 129 is wherein passed through: the one or more methods in T-DNA activation, TILLING and the homologous recombination realize described genetic modification.

131. be used for comparing the method that increases monocotyledonous seed production with the seed production of suitable control plant, this method is included in and imports and preferentially express therein the nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain in the monocotyledonous endosperm tissue.

132. the method for claim 131, wherein said polypeptide also comprise a primitive in the following primitive: primitive 1:QGQ V/I GG; Or primitive 2:ILSLSGSFLPPPAPP; Or primitive 3:NATYERLP; Or primitive 4:SFTNVAYERLPL, have 0 or 1 amino acid and change in any position; Or primitive 5:GRFEILSLTGSFLPGPAPPGSTGLTIYLAGGQGQVVGGSVVG, have 0,1 or 2 amino acid and change in any position.

133. the method for claim 131 or 132, wherein said nucleic acid and endosperm specificity promoter, preferred prolamine promotor effectively connects.

134. the method for each of claim 127 to 133, wherein said nucleic acid is SEQ IDNO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:156, SEQID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, part or the allele variant or the splice variant of any one sequence among SEQ ID NO:168 and the SEQ ID NO:170, or can with the sequence of described sequence hybridization, wherein said part, allele variant, splice variant or hybridization sequences coding comprise the polypeptide of AT hook structure territory and DUF296 structural domain.

135. the method for claim 134, proteic lineal homologue of AT hook or the collateral line homologue of wherein said part, allele variant, splice variant or hybridization sequences coding SEQ ID NO:153.

136. the method for each of claim 127 to 135, the nucleic acid that wherein said coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain is plant origin, preferably from monocotyledons, further preferably from Gramineae, more preferably from Oryza, most preferably from rice.

137. the method for each of claim 127 to 136, the output of wherein said increase is selected from: in the number of the seed sum of the seed gross weight of increase, the full seed number of increase, increase, every paniculiform flower of increase, the harvest index (HI) of increase one or multinomial.

138. plant or its part that can obtain by 127 to 137 each method, comprise vegetable cell, wherein said plant or its part comprise the nucleic acid that coding contains the polypeptide of AT hook structure territory and DUF296 structural domain, and this nucleic acid effectively is connected with endosperm specificity promoter.

139. a gene construct, it comprises:

(a) coding comprises the nucleic acid of the polypeptide of AT hook structure territory and DUF296 structural domain;

(b) can in monocotyledonous endosperm tissue, drive one or more control sequences that the nucleotide sequence of (i) is expressed; Randomly

(c) transcription termination sequence.

140. the construct of claim 139 is used for increasing in monocotyledons the purposes of seed production.

141. the construct of claim 139, wherein said control sequence are the prolamine promotors.

142. use construct plant transformed, plant part or the vegetable cell of claim 139.

143. be used to produce the monocotyledonous method of transgenosis of comparing the seed production with increase with suitable control plant, this method comprises:

(a) in monocotyledonous endosperm tissue, import and preferentially express therein the nucleic acid that coding comprises the polypeptide of AT hook structure territory and DUF296 structural domain; With

144 compare the transgenosis monocotyledons of the seed production with increase with suitable control plant, the seed production of described increase is because coding comprises the nucleic acid of the polypeptide of AT hook structure territory and DUF296 structural domain preferentially expresses in monocotyledonous endosperm tissue and cause.

145. claim 138,142 or 144 transgenosis monocotyledons, wherein said plant is a cereal, for example rice, corn, sugarcane, wheat, barley, grain, rye, Chinese sorghum, grass or oat.

146. claim 138,142,144 or 145 each the part gathered in the crops of plant, wherein said to gather in the crops part be seed.

147. derive from the plant of claim 145 and/or derive from the product of the part gathered in the crops of the plant of claim 146.

148. coding comprises the nucleic acid of the AT hook structure territory and the polypeptide of DUF296 structural domain and is used for comparing the purposes that increases monocotyledonous seed production with the seed production of suitable control plant, described nucleic acid effectively is connected with endosperm specificity promoter.

149. the purposes of claim 148, the seed production of wherein said increase is selected from: in the number of the seed sum of the seed gross weight of increase, the full seed number of increase, increase, every paniculiform flower of increase, the harvest index (HI) of increase one or multinomial.

150. be used for comparing the method that increases plant biomass with suitable control plant, this method is included in increases encoding D OF (DNA-binding with one finger in the plant, singly to refer in conjunction with DNA) expression of nucleic acids of structural domain transcription factor polypeptide, described polypeptide comprises following feature (i) and also comprises following feature (ii) or (iii):

(i) with the DOF structural domain of representing by SEQ ID NO:200 or SEQ ID NO:228 at least 60% sequence identity is arranged; With

At least 70% sequence identity is (ii) arranged with the DOF structural domain of representing by SEQ ID NO:200; Or

151. the method for claim 150 wherein comprises feature (i) and feature described polypeptide (iii) and also comprises any one in the following primitive, any two or all three primitives:

-primitive III: unconverted or have that one or more conservative propertys in any position change or have one, two or three SPTLGKHSRDE (SEQ ID NO:231) that change at the non-conservation of any position; And/or

152. the method for claim 150 or 151 wherein comprises feature (i) and feature described polypeptide (iii) and comprises primitive I and II.

153. the method for each of claim 150 to 152 is wherein passed through, and preferably in the locus of the gene of encoding D OF transcription factor polypeptide, imports the expression that genetic modification is realized described increase.

154. the method for claim 153 is wherein passed through: T-DNA activation, TILLING and homologous recombination realize described genetic modification.

155. be used for comparing the method that increases plant biomass with suitable control plant, this method is included in the plant nucleic acid or its variant that imports and express encoding D OF transcription factor polypeptide, and wherein said polypeptide comprises following feature (i) and also comprises following feature (ii) or (iii):

156. the method for claim 155 wherein comprises feature (i) and feature described polypeptide (iii) and also comprises any one in the following primitive, any two or all three primitives:

157. the method for claim 155 or 156 wherein comprises feature (i) and feature described polypeptide (iii) and comprises primitive I and II.

158. the method for claim 155 to 157, the nucleic acid of wherein said encoding D OF transcription factor or its variant are crossed in plant and are expressed.

159. the method for each of claim 155 to 158, wherein said nucleic acid or its variant are plant origins, and preferably from dicotyledons, more preferably from Cruciferae, more preferably this nucleic acid is from Arabidopis thaliana.

160. the method for each of claim 155 to 159, the homologue of the DOF transcription factor protein of wherein said variant coding SEQID NO:199 or SEQ ID NO:227.

161. the method for claim 160, the homologue of the DOF transcription factor protein of wherein said SEQ ID NO:199 is by any expression among SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220 and the SEQ IDNO:222.

162. the method for claim 160, the homologue of the DOF transcription factor protein of wherein said SEQ ID NO:227 is by any expression among SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253 and the SEQ IDNO:255.

163. the method for each of claim 155 to 162, nucleic acid or its variant of wherein said encoding D OF transcription factor polypeptide effectively are connected with constitutive promoter.

164. the method for claim 163, wherein said constitutive promoter are the GOS2 promotors, preferably from the GOS2 promotor of rice.

165. the method for each of claim 155 to 162, nucleic acid or its variant of wherein said encoding D OF transcription factor polypeptide effectively are connected with seed specific promoters.

166. the method for claim 165, wherein said seed specific promoters is an endosperm specificity promoter, preferred prolamine promotor.

167. the method for claim 163 or 164, wherein said constitutive promoter drives the expression of nucleic acids that coding comprises described feature (i) and DOF transcription factor polypeptide (ii).

168. the method for claim 165 or 166, wherein said seed specific promoters drives the expression of nucleic acids that coding comprises described feature (i) and DOF transcription factor polypeptide (iii).

169. the method for each of claim 150 to 168, the output of wherein said increase is selected from: in the root length of the harvest index (HI) of the number of every paniculiform flower of the full seed number of increase, the seed weight of increase, increase, the full rate of seed of increase, increase, the thousand seed weight (TKW) that increases, the root biomass that increases, increase and the root diameter of increase one or multinomial.

170. the method for each of claim 150 to 169, the output of wherein said increase takes place under slight drought stress.

171. plant or its part that can obtain by each method of claim 150 to 170.

172. a construct, it comprises:

(i) nucleic acid or its variant of the DOF transcription factor polypeptide of definition in the coding claim 155;

(ii) can drive one or more control sequences of the nucleotide sequence expression of (a); Randomly

(iii) transcription termination sequence.

173. the construct of claim 172, wherein said control sequence is a constitutive promoter.

174. the construct of claim 172, wherein said control sequence is a seed specific promoters.

175. each construct plant transformed, plant part or vegetable cell with claim 172 to 174.

176. be used to produce the method for comparing the transgenic plant of the output with increase with corresponding wild type plant, this method comprises:

(a) nucleic acid or its variant of the DOF transcription factor polypeptide that in plant, plant part or vegetable cell, defines in importing and the expression coding claim 155;

177. the method for claim 176, the output of wherein said increase takes place under the condition of slight drought stress.

178. compare the transgenic plant of the output with increase with corresponding wild type plant, the output of described increase is because nucleic acid or its variant of the DOF transcription factor polypeptide of the coding claim 155 of the described plant of importing cause.

179. claim 171,175 or 178 transgenic plant, wherein said plant is a monocotyledons, for example sugarcane or wherein this plant be cereal, for example rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum.

180. claim 171,175,178 or 179 each the part gathered in the crops of plant.

181. the part gathered in the crops of the plant of claim 180, wherein said gather in the crops the part be seed.

182. derive from the plant of claim 179 and/or derive from the product of the part gathered in the crops of the plant of claim 180 or 181.

183. transcribing because of the nucleic acid of polypeptide or its variant or DOF transcribe because of polypeptide, encoding D OF is used for comparing the purposes that improves plant biomass with suitable control plant.

184. the purposes of claim 183, the output of wherein said increase is selected from: in the root length of the harvest index (HI) of the number of every paniculiform flower of the full seed number of increase, the seed weight of increase, increase, the full rate of seed of increase, increase, the thousand seed weight (TKW) that increases, the root biomass that increases, increase and the root diameter of increase one or multinomial.

185. the purposes of claim 183 or 184, the output of wherein said increase takes place under slight drought stress condition.

186. encoding D OF transcribes because of the nucleic acid of polypeptide or its variant or DOF and transcribes because of the purposes of polypeptide as molecule marker.

187. be used for comparing with suitable control plant the method for the seed production that increases plant, this method comprises the preferentially endogenous CKI expression of gene of minimizing in the endosperm tissue of plant.

188. the method for claim 187, wherein the down regulation of gene expression by the RNA mediation realizes described preferential minimizing expression.

189. the method for claim 188 is wherein by suppressing to realize the downward modulation of described RNA mediation altogether.

190. the method for claim 188 is wherein by using antisense CKI nucleotide sequence to realize the downward modulation of described RNA mediation.

191. the method for claim 187, wherein by CKI gene or its segmental inverted repeats, the inverted repeats that preferably can form hairpin structure is realized described preferential minimizing expression.

192. the method for claim 187 is wherein used for the specific ribozyme of CKI nucleic acid tool and is realized described preferential minimizing expression.

193. the method for claim 187 wherein realizes described preferential minimizing expression by inserting mutagenesis.

194. the method for each of claim 187 or 193, wherein by endosperm specificity promoter, preferred prolamine promotor realizes the preferential minimizing expression of described endogenous CKI gene in the endosperm tissue of plant.

195. the method for each of claim 187 to 194, wherein said endogenous CKI gene are to be present in the CKI gene in the plant or to import the isolating CKI gene of plant subsequently with its natural form.

196. the method for each of claim 187 to 197, wherein said CKI gene/nucleic acid is from plant origin or artificial source, preferably wherein be used for transforming monocots from monocotyledonous CKI nucleotide sequence, more preferably wherein be used for transforming gramineous plant, more preferably wherein be used to transform rice plant from the CKI nucleotide sequence of rice from CKI sequence gramineous.

197. the method for claim 196, wherein comprise the Nucleotide of successive basically of sufficient length among the SEQ ID NO:267 (OsCKI4) from the described CKI nucleotide sequence of rice comprise the lineal homologue of coding OsCKI4 (SEQ ID NO:267) or the nucleotide sequence of collateral line homologue in the Nucleotide of successive basically of sufficient length.

198. the method for claim 197, the lineal homologue of wherein said OsCKI4 or collateral line homologue are represented by SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQID NO:276, SEQ ID NO:278 and SEQ ID NO:280.

199. the method for claim 197 or 198, in the lineal homologue of wherein said coding OsCKI4 (SEQ IDNO:267) or the nucleotide sequence of collateral line homologue basically successive Nucleotide be: the Nucleotide of successive basically that comes the nucleotide sequence that free SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277 or SEQ ID NO:279 represent.

200. the method for each of claim 187 to 199, the seed production of wherein said increase is selected from: a) the seed biomass of Zeng Jiaing; B) number of the flower of every strain plant of Zeng Jiaing; C) (full) number seeds of Zeng Jiaing; And d) in the harvest index of Zeng Jiaing or multinomial.

The method of each of claim 187 to 200, the output of wherein said increase takes place under slight stress conditions.

Plant or its part that can obtain by each method of claim 187 to 201.

Be used to produce the method for comparing the transgenic plant of the seed production with increase with suitable control plant, this method comprises:

(a) in plant, plant part or vegetable cell, import and express the gene construct that comprises one or more control sequences, described control sequence can drive justice and/or antisense CKI nucleotide sequence is preferentially expressed in the albumen tissue, with reticent endogenous CKI gene in the endosperm tissue of plant; With

(b) culturing plants, plant part or vegetable cell under the condition that promotes plant-growth and growth.

CKI nucleic acid is used in the minimizing of albumen tissue or eliminates endogenous CKI genetic expression in fact to compare the purposes that increases the seed production in the plant with suitable control plant.

The purposes of claim 204, the output of wherein said increase is selected from: a) the seed biomass of Zeng Jiaing; B) quantity of the flower of every strain plant of Zeng Jiaing; C) (full) number seeds of Zeng Jiaing; And d) in the harvest index of Zeng Jiaing or multinomial.

Claim 204 or 205 purposes, wherein said seed production is producing under slight stress conditions.