CN108424918A

CN108424918A - It is a kind of inhibit wheaten starch synthesis transcription factor WSR1 genes and its application

Info

Publication number: CN108424918A
Application number: CN201810301494.5A
Authority: CN
Inventors: 吕亮杰; 张春风; 李辉; 陈希勇; 宿振起; 孙丽静; 王莉梅
Original assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Current assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Priority date: 2018-04-04
Filing date: 2018-04-04
Publication date: 2018-08-21

Abstract

The invention discloses a kind of transcription factor WSR1 genes of inhibition wheaten starch synthesis and its application, which includes a) or b) any nucleotide sequence：A) nucleotide sequence shown in SEQ ID NO.1；B) nucleotide sequence shown in SEQ ID NO.1 is through replacing, the nucleotide sequence with same function of missing or addition base formation.SEQ ID NO are overexpressed in wheat：Gene shown in 1 inhibits SEQ ID NO：The expression of gene shown in 1 is of great significance with cultivating the transgenic wheat with high-content of starch to improving wheat yield.

Description

It is a kind of inhibit wheaten starch synthesis transcription factor WSR1 genes and its application

Technical field

The invention belongs to biology and gene engineering technology field, specifically, be related to it is a kind of inhibition wheaten starch synthesis Transcription factor WSR1 genes and its application.

Background technology

Starch is the main component of wheat seed, accounts for about 65% or so of wheat seed weight, content of starch and straight, branch Shallow lake content has a major impact wheat quality.Wheat is the second largest grain of one of most important cereal crops and China in the world Food crop.The biosynthesis of starch is a complicated process, is influenced by various synthesis key enzymes, while environment closes starch At also there is large effect.The molecular mechanism for studying wheaten starch synthesis, it is to improve wheat production to cultivate high starch new variety of wheat One main path of amount, for ensureing that national food security is of great significance.Some scholars have studied and starch in recent years The relevant transcription factor of route of synthesis.

Starch synthesis is a complicated biological process in plant cell, and some scholars have studied and Starch synthesis in recent years The relevant transcription factor of approach.Transcription factor (transcription factor) refers to can be with modulated target gene 5 ' end Upstream sequence combines, and regulates and controls the protein of target gene spatial and temporal expression function.Because the position that it is combined with DNA sequence dna is referred to as cis- Functional element (cis-actingelement) is combined, so transcription factor is also referred to as trans-acting factor.Zhu (Zhu Y, Hong MM, Wang ZY.Identification of the Regions Involved in Protein-Protein Interaction of OsBP-5 and OsEBP-89[J],Acta Photophysiologica Sinica,2003,29 (5):425-430) etc. by EREBP albumen (ethylene responsive element binding protein) the study found that EREBP can improve Waxy The transcriptional level of (GBSS granule bound starch synzyme) gene.Rook (Rook F, Corke F, Card R, et al.Impaired sucrose-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signaling[J].Plant Journal for Cell&Molecular Biology,2001,26(4):421-433) etc. find apetala2-type albumen The transcriptional level of AGPL3 (glucose pyrophosphorylase large subunit) can be improved.(Fu F F, the Xue H such as Fu W.Coexpression analysis identifies rice starch regulator1,a rice AP2/EREBP family transcription factor,as a novel rice starch biosynthesis regulator[J] .Plant Physiol.2010,154:927-938.) by co-expressing analysis and identification one negative regulation, 15 rice feculas The Starch synthesis regulative transcription factor (Rice Starch Regulatory RSR1) of synthesis related gene expression.To pass through base Because engineering or other methods improve the accumulation of wheaten starch, improving quality, raising grain have established solid foundation again.

The report of transcription factor is synthesized almost without majority only guesses its function, fails to current research wheaten starch Effectively grind card.

Invention content

In view of this, the present invention provides a kind of transcription factor WSR1 genes of inhibition wheaten starch synthesis and its applications.

In order to solve the above-mentioned technical problem, the invention discloses a kind of transcription factor WSR1 bases of inhibition wheaten starch synthesis Cause, including a) or b) any nucleotide sequence：

A) nucleotide sequence shown in SEQ ID NO.1；

B) nucleotide sequence shown in SEQ ID NO.1 lacks through replacing or what addition base was formed has same function Nucleotide sequence.

The invention also discloses a kind of albumen of the above-mentioned transcription factor WSR1 gene codes for inhibiting wheaten starch synthesis.

Optionally, amino acid is as shown in SEQ ID NO.7.

The transcription factor for inhibiting wheaten starch synthesis containing above-mentioned nucleotide sequence that the invention also discloses a kind of WSR1 expression vectors.

The invention also discloses a kind of host cells containing above-mentioned expression vector.

The invention also discloses a kind of above-mentioned transcription factor WSR1 genes for inhibiting wheaten starch synthesis to cultivate high form sediment Application in powder genetically modified plants.

Optionally, the high starch genetically modified plants are wheat.

The invention also discloses a kind of breeding methods of the transgenic wheat with high starch, and SEQ is overexpressed in wheat ID NO：Gene shown in 1 inhibits SEQ ID NO：The expression of gene shown in 1, it is small to cultivate the transgenosis with high starch Wheat.

The invention also discloses a kind of above-mentioned transcription factor WSR1 genes for inhibiting wheaten starch synthesis to cultivate high yield Measure the application in genetically modified plants.

Optionally, the high yield genetically modified plants are wheat.

Compared with prior art, the present invention can be obtained including following technique effect：

The present invention provides a new wheat cdna, real-time fluorescence quantitative PCR testing result shows gene of the present invention Expression affects the content of starch synthesis of wheat, illustrates that it plays an important role in wheaten starch synthesis mechanism, is based on this, can Using its transgenic wheat of the cultivation with high-content of starch, it is of great significance to improving wheat yield.

Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technique effect.

Description of the drawings

Attached drawing described herein is used to provide further understanding of the present invention, and constitutes the part of the present invention, this hair Bright illustrative embodiments and their description are not constituted improper limitations of the present invention for explaining the present invention.In the accompanying drawings：

Fig. 1 is WSR1 gene structures figure of the present invention, and box represents exon, and lines represent introne, and arrow represents non-volume Code area, pentagon represent conserved domain；

Fig. 2 is that the present invention carries out homology ratio using DNAMAN softwares to wheat WSR1 and rice Os UreG amino acid sequences To result；

Fig. 3 is that Ji wheat 325 of the present invention is inoculated with BSMV:The wheat leaf blade situation of change of PDS：Wherein, BSMV:00 is control group It is not inoculated with BSMV:PDS 15 days wheat leaf blades of virus；BSMV:PDS is inoculation BSMV:The wheat leaf blade of PDS viruses, 5d, 10d, 20d respectively represents the wheat leaf blade after being inoculated with 5 days, 20 days 10 days；

Fig. 4 is real-time fluorescence quantitative PCR of the present invention to silence PDS genes and silence WSR1 genes when Ji wheat 325 is different Between lower expression characteristic analysis result, BSW:WSR1 is the expression of results for being inoculated with WSR1 gene different times；

Fig. 5 is amylose content (AAC), the branch of present invention control Ji wheat 325 and the Ji wheat 325 of silence WSR1 genes Content of starch (APC), total starch content (SC), resistance starch content (RS) variation diagram；Wherein, WT is control Ji wheat 325, WSR1 For the Ji wheat 325 of silence WSR1 genes；

Fig. 6 is mass of 1000 kernel (Fig. 6 A) and the Grain Filling of present invention control Ji wheat 325 and the Ji wheat 325 of silence WSR1 genes As a result (Fig. 6 B)；WT is control Ji wheat 325, and WSR1 is the Ji wheat 325 of silence WSR1 genes.

Specific implementation mode

Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.

Gene source provided by the invention is named as WSR1 (Wheat in wheat Ji wheat 325 according to sequence structure feature Starch Regulatory 1), cDNA sequence overall length 2003bp includes the open reading frame of an a length of 1584bp, coding 527 amino acid；Genome DAN sequence 4316bp, are made of 8 exons and 7 intrones.Since the ends 5', outside The length for showing son is followed successively by 1070bp, 58bp, 88bp, 146bp, 104bp, 132bp, 109bp, 585bp, the length of introne according to Secondary is 121bp, 121bp, 136bp, 490bp, 126bp, 367bp, 945bp, and gene structure is referring to Fig. 1.

Embodiment 1：The clone for inhibiting the transcription factor gene WSR1 of wheaten starch synthesis is Ji wheat 325 using material.

1. wheat WSR1 cDNA sequence electronic clonings；

(1) rice RSR1 sequence (the GenBank accession number submitted with NCBI:LOC_Os05g03040 wheat cdna) is compared Group sequence designs the primer of electronic cloning to obtain homologous fragment as target sequence, by SMARTTM RACE cDNA Requirement in Amplification Kit designs reversed special primer WSR1-5 using 5.0 softwares of Primer at its end 5', Positive special primer WSR1-3 is designed at its end 3', obtained sequence DNAMAN softwares are spliced, to open reading frame (ORF) It is predicted, to obtain the cDNA sequence of complete wheat WSR1.

(2) according to obtained full length cDNA sequence Primer5 Software for Design and a pair of of special primer WSR1-F is filtered out And WSR1-R, it is used for wheat WSR1 cDNA and gDNA gene clonings.

Forward primer WSR1-F：5’-TCTTCTTGAATCGTGAGGTT-3’(SEQ ID NO：3)；

Reverse primer WSR1-R：5’-GCTGAGGTTAGTATCTGACA-3’(SEQ ID NO：4)；

2. extracting total serum IgE using Trizol (Invitrogen), it is as follows：

(1) liquid nitrogen grinding 50-100mg materials are transferred to after grinding in the EP pipes of the reagents of Trizol containing 1ml, fully mixed It is even, it is stored at room temperature 5min.

(2) 0.2ml chloroforms are added, mixes well, is stored at room temperature 2-3min；12000g, centrifuges 15min by 4 DEG C.

(3) the colourless water phase in upper layer is transferred in a new 1.5ml EP pipe, addition 0.5ml isopropanols, after mixing It is placed at room temperature for 10min；12000g, centrifuges 10min by 4 DEG C.

(4) supernatant is removed, RNA precipitate is cleaned with 75% ethyl alcohol of 1ml, 7500g, 4 DEG C, 5min is centrifuged, air-dries later.

(5) precipitation is dissolved in the deionized water of appropriate RNase-free, 60 DEG C of dissolution 10min.

(6) micro-spectrophotometer NanoDrop ND-2000 Spectrotometer are quantitative, and electrophoretic analysis RNA is complete Property.

3. extracting DNA using plant genome DNA extracts kit (Tiangeng, DP305), it is as follows：

(1) fresh tissues of plants about 100mg is taken, liquid nitrogen is added and fully mills.

(2) ground powder is quickly transferred in the centrifuge tube for being pre-loaded with 700 μ l, 65 DEG C of preheating buffer solution GP1 (mercaptoethanol is added in the GP1 of preheating before experiment, keeps its final concentration of 0.1%), rapidly after reverse mixing, centrifuge tube is put Reverse centrifuge tube is to mix sample for several times during 65 DEG C of water-bath 20min, water-bath.

(3) 700 μ l chloroforms are added, mix well, 12000rpm centrifuges 5min.

(4) carefully upper strata aqueous phase obtained by previous step is transferred in a new centrifuge tube, 700 μ l buffer solution GP2 is added, It mixes well.

(5) liquid of mixing is transferred in adsorption column CB3,12000rpm centrifuges 30s, discards waste liquid.

(6) 500 μ l buffer solutions GD, 12000rpm centrifugation 30s are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 is put into collecting pipe.

(7) 600 μ l rinsing liquids PW, 12000rpm centrifugation 30s are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3 is put into collecting pipe.

(8) repetitive operation step 7.

(9) adsorption column CB3 is put back in collecting pipe, 12000rpm centrifuges 2min, outwells waste liquid.Adsorption column CB3 is placed in It is placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.

(10) adsorption column CB3 is transferred in a clean centrifuge tube, 50- is vacantly added dropwise to the intermediate position of adsorbed film 200 μ l elution buffer TE, are placed at room temperature for 2-5min, and 12000rpm centrifuges 2min, solution is collected into centrifuge tube.

4. utilize GoldScript cDNA synthetic agent box (Invitrogen, c81401190)) carry out the first chain cDNA Synthesis, is as follows：

(1) 5 μ l of total serum IgE (200ng/ μ l), 10mM dNTP mix 1 μ l, Oligo are sequentially added in 0.2ml centrifuge tubes 1 μ l, DEPC water of dT Primer, 3 μ l.

(2) above-mentioned RNA/ primer mixtures are placed on 65 DEG C of incubation 5min, are then placed at least 1min on ice.

(3) in another pipe, 2 × reaction mixture is prepared, following component is sequentially added into：10 × RT buffer solutions, 2 μ l, 25mM MgCl₂4 μ l, 0.1M DTT, 2 μ l recombinate RNase inhibitor (40U/ μ l) 1 μ l.

(4) be added 9 μ 2 × reaction premixed liquids of l into each RNA/ primers premixed liquid of the 2nd step, soft mixing, it is of short duration from The heart.

(5) 42 DEG C of incubation 2min.

(6) 1 μ l GoldScript RT are added in each pipe.

(7) 42 DEG C of incubation 50min.

(8) 70 DEG C of incubation 15min, terminate reaction, cooled on ice.

(9) of short duration that reaction is collected by centrifugation, 1 μ l RNaseH are added into each pipe, 37 DEG C of incubation 20min, -20 DEG C preserve.

5. PCR (PCR) reaction carries out sequence amplification

PCR amplification, PCR amplification system 50 are carried out to wheat cDNA and gDNA respectively with WSR1-F and WSR1-R primers 10 μ l, dNTP Mixture of μ l, 5 × PrimeSTAR GXL Buffer 4 μ l, Forward Primer (10 μM) 2.5 μ l, 2 μ l, PrimeSTAR GXL DNA Polymerase of Reverse Primer (10 μM) 2.5 μ l, template cDNA or gDNA (TaKaRa, R050Q) 0.5 μ l, ddH₂O 28.5μl.PCR response procedures：98℃2min；98 DEG C of 10s, 58 DEG C of 15s, 68 DEG C 3min, 30 cycles；68℃10min；4 DEG C of preservations.

6.PCR products carry out purifying recycling using Ago-Gel QIAquick Gel Extraction Kit (Tiangeng, DP209), and specific steps are such as Under：

(1) column equilibration step：Into adsorption column CA2,500 μ l equilibrium liquid BL are added in (adsorption column is put into collecting pipe), 12000rpm centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

(2) single target DNA band is put into clean centrifuge tube from being cut in Ago-Gel, weighs weight.

(3) equimultiple bulk solution PN is added into blob of viscose, 50 DEG C of water-baths are placed, and constantly leniently spin upside down centrifugation therebetween Pipe, to ensure that blob of viscose fully dissolves.

(4) previous step acquired solution is added in an adsorption column CA2 (adsorption column is put into collecting pipe), is placed at room temperature for 2min, 12000rpm centrifuge 30-60s, outwell the waste liquid in collecting pipe, adsorption column CA2 is put into collecting pipe.

(5) 600 μ l rinsing liquids PW, 12000rpm are added into adsorption column CA2 and centrifuge 30-60s, outwell useless in collecting pipe Adsorption column CA2 is put into collecting pipe by liquid.

(6) repetitive operation step 5.

(7) adsorption column CA2 is put back in collecting pipe, 12000rpm centrifuges 2min, eliminates rinsing liquid as possible.By adsorption column CA2, which is placed in, to be placed at room temperature for several minutes, is thoroughly dried.

(8) adsorption column CA2 is put into a clean centrifuge tube, it is slow that appropriate elution is vacantly added dropwise to adsorbed film centre position Fliud flushing EB, is placed at room temperature for 2min.12000rpm centrifuges 2min and collects DNA solution.

7. gene cloning：Take 4 μ l PCR recovery products be added 1 μ l pEASY-Blunt Cloning carriers (Quan Shijin, CB101-01), it is gently mixed, (20 DEG C -37 DEG C) reaction 5min of room temperature complete connection；Connection product is converted into bacillus coli DH 5 α bacterial strains are coated with 8 μ l IPTG (500mM), kanamycins (50 μ g/ml) LB tablets of 40 μ l X-gal (20mg/ml) on surface 37 DEG C of growths are overnight；White colony is selected, positive colony sequencing is selected by fast PCR.

8. wheat WSR1 sequence analyses

(1) sequencing result is analyzed with DNAMAN softwares, with the wheat of WSR1-F and WSR1-R primer amplifications The cDNA sequence of WSR1 such as SEQ ID NO：Shown in 1, cDNA sequence overall length 2003bp includes the opening of an a length of 1584bp Reading frame encodes 527 amino acid.With the genomic dna sequence such as SEQ of the wheat WSR1 of WSR1-F and WSR1-R primer amplifications ID NO：Shown in 2, gDNA sequence 4763bp, including 8 exons and 7 intrones.Since holding 5 ', exon Length is followed successively by 1070bp, 58bp, 88bp, 146bp, 104bp, 132bp, 109bp, 585bp, and the length of introne is followed successively by 121bp, 121bp, 136bp, 490bp, 126bp, 367bp, 945bp, gene structure is referring to Fig. 1.

(2) wheat WSR1 and the detection of rice RSR1 amino acid identities are detected.The amino acid sequence of wheat WSR1 such as SEQ ID NO：Shown in 7.The amino acid sequence of wheat WSR1 and rice RSR1 amino acid sequences are subjected to sequence with DNAMAN softwares It compares, the two homology is 53.99%, and comparison result is shown in Fig. 2.

The VIGS silence wheats WSR1 of embodiment 2

After wheat flower 12 days, the wheat Ji wheat 325 of robust growth is chosen.By the good elimination of barley's yellow mosaic of in-vitro transcription Malicious α, β and γ transcription product is added the DEPC water of 2 times of volumes (60 μ L), adds the 1 of ingredient at this time after respectively taking 10 μ L mixing The mixed liquor for the virus inoculation that total volume is 210 μ L is made in the GKP Buffer of times volume (120 μ L).It to wear when inoculation and just open Rubber gloves is sealed, takes 10 μ L inoculation liquids to be placed on index finger tripe with the water-treated pipette tips of DEPC, a hand fixes the wheat wheat head One end, the other hand thumb and index finger gently back and forth rub the wheat wheat head.After being inoculated with, sprayed to wheat seedling after 20min DEPC water, moisturizing is for 24 hours.It observed daily after inoculation, record inoculation position variation, and periodically drawn materials.It is inoculated with BSMV:The wheat of PDS There is bleaching phenomenon in blade, and be only inoculated with wheat WSR1 genes does not have significant reaction.As a result such as Fig. 3, inoculation BSMV is found:PDS's Wheatear started photobleaching phenomenon occur at the 5th day, and at the 10th day, photobleaching phenomenon was obvious, was floated the 20th day time Reach utmostly in vain, and is inoculated with BSMV:00 control group is but without significant change, and rate of vaccination is up to 90%.

3 real-time fluorescence quantitative PCR of embodiment (Real-time PCR) verifies works of the WSR1 in wheaten starch synthesis mechanism With

1. material selection

It chooses the Ji wheat 325 of Field inoculation silence WSR1 genes, 5d, 10d, 15d, 20d, 25d and is not inoculated with 325 and takes respectively Sample, -80 DEG C of preservations after liquid nitrogen frozen.

2. extracting total serum IgE using Trizol, specific steps are referring to embodiment 1.

3. utilizing PrimeScript^TMRT reagent Kit with gDNA Eraser (TaKaRa, RR047A) are carried out First chain cDNA synthesis：

(1) genomic DNA, reaction system are removed：5 × gDNA Eraser Buffer, 2 μ l, gDNA Eraser, 1 μ l, 5 μ l of Total RNA (200ng/ μ l), RNase Free dH₂O 2μl；Response procedures：42 DEG C of 2min, 4 DEG C of preservations.

(2) reverse transcription, reaction system：10 μ l, PrimeScript RT Enzyme Mix I of reaction solution, the 1 μ l of step 1, 1 μ l, 5 × PrimeScript Buffer of RT Primer Mix, 24 μ l, RNase Free dH₂O 4μl；Response procedures：37 DEG C 15min, 85 DEG C of 5s, 4 DEG C of preservations.

4. usingPremix Ex Taq II (TaKaRa, RR820A) kit carries out real-time fluorescence quantitative PCR Analysis

Then the following reaction system of mixing is sub-packed in 96 hole optics versions, and cover upper optical film, expands the instrument used For ABI PRISM 7500real-time PCR instruments；

PCR response procedures：95℃30s；95 DEG C of 5s, 60 DEG C of 34s, 40Cycles；95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s；

20 μ l PCR reaction systems are formulated as follows：Premix Ex Taq II (2 ×) 10 μ l, Forward 0.8 μ l, Reverse Primer (10 μM) of Primer (10 μM) 0.8 μ l, ROX Reference Dye II (50 ×) 0.4 μ l, CDNA templates 2 μ l, ddH₂O 6μl；

Primer sequence is：WSR1 special primers, WSR1-F：5’-TGAAGTTGCTGCTGAAGG-3’(SEQ ID NO：5), WSR1-R：5’-CGATGACGACGAGAAGAG-3’(SEQ ID NO：6).

5. result

Referring to Fig. 4, the results show that being vaccinated with BSMV:After PDS, the relative expression quantity of wheat fringe portion PDS genes is apparent It reduces, has dropped 50% within the 5th day after inoculation, 73% was had dropped at the 15th day and is continued until the 25th day；It is being vaccinated with BSMV:After WSR1, the relative expression quantity of wheat fringe portion WSR1 genes significantly reduces, and has dropped 37% within the 5th day after inoculation, 75% was had dropped at the 15th day to be continued until the 25th day, had dropped 88%.Fig. 5 is the results show that be vaccinated with BSMV:WSR1 it Afterwards, straight chain, branch, total starch, resistance starch content are above nonvaccinated Ji wheat 325.Fig. 6 is the results show that being vaccinated with BSMV:After WSR1,325 seed mass of 1000 kernel of wheat Ji wheat and volume are significantly greater than nonvaccinated 325 seed of Ji wheat.Illustrate WSR1 It plays an important role in inhibiting wheaten starch synthesis mechanism, WSR1 is overexpressed in wheat or inhibits the expression of WSR1, will influence small The transgenic wheat with high starch is cultivated in the synthesis of wheat starch, is of great significance to improving wheat yield.

Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not It is confined to form disclosed herein, is not to be taken as excluding other embodiments, and can be used for various other combinations, modification And environment, and can be carried out by the above teachings or related fields of technology or knowledge in the scope of the invention is set forth herein Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then should all be weighed appended by invention In the protection domain that profit requires.

Sequence table

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cgccggcggc ggtccgccgc cgatgcccgt gccgcagcat tgggccgagc tcggcttctt 900

ccgccccgcg gcgccgcccc cggacatgag gatcctgcag ctgcagcagc aggcgcacgc 960

gccgcccccg ccgccgccgg cggcccagcc gccggtggcc aagaagagcc gccgcggccc 1020

gcgctcccgc agctcgcagt accgcggcgt gaccttctac cgccgcaccg gccgctggga 1080

atcccatatc tggtccgtac acaaatccct ccaaccaccc ccaaaaagaa acccaatttt 1140

tttcctcaaa tatcaccaac tgattgtttc agttcttaca aattttcttg ctcctgttca 1200

tcgctaaatg cagggattgc ggcaagcagg tgtacttggg tggatttgac actgcacatg 1260

ctgctgcaag ggtacaaatt taattaagca cgagtacata attgtgatgt gatcatcacc 1320

tgaactacct gtactgaaac cctcaagtca tgtcatttca ccgtgccaaa ttgaccttgg 1380

gatgttccgc agggcgtacg atcgagcggc gatcaagttc cgtggcgtcg acgccgacat 1440

aaacttcaac ctcagcgact acgaggacga catgaagcag gtgatcggca aagccaccaa 1500

ccagtgttcc tcatccaacc aaatagttca gatgcagagt gcattagtac tgttgttgaa 1560

actgatgaac tgaagaaatt ctgactgtgt gttgtttggt ggatgatctg gatcagatga 1620

agggcctgtc caaggaggag ttcgtgcacg tgctgcggcg gcagagcacc ggcttctcgc 1680

ggggcagctc caagtacaga ggcgtcaccc tgcacaagtg cggccggtgg gaggcgcgca 1740

tgggccagtt cctcggcaag aagtaagaac aaattcccct ttccattttg aaccgatttt 1800

tcatttctta cttcatggca tgttgcactg aatgcacttg tgaagttaac atatgcatct 1860

ctggatgtga ttgtgctgct gcctatctga tctgaatctg aatctcgtaa gagagagtca 1920

tgatcagatc aaacaagatc ccatgatcca ttaggaatgt attttaagca gtagttcata 1980

ggttgaaaat actaagatgc atagatcagg aaaaataaaa tgaaatagta gtaggcagtg 2040

gatagggagt tcctcaaacg acatgtttgg gtgcaggtac atatatcttg ggctattcga 2100

caatgaagta gaggctgcaa ggttcctgag cttggattct gcccattgat gcacaataaa 2160

aaagttgttt cttttttctc catcaaccga agtaccaact cgaatctctt ctctttgttt 2220

ctcttcttct ttttcttgtc cggaaaaatc agggcttacg acaaggcggc gatcaaatgc 2280

aacggtaggg aggccgtgac gaacttcgag ccgagcacct atgatgcgga gctgctcagt 2340

gaggttgctg ctgaaggtaa caacatgatg agaattttga ttgaatttat ttttcatgtt 2400

tgcatctgtg tatctgtaat ttggcaaaat tctgcgttgg agattagcta atgaacttca 2460

atttgattat gacgattggc aggcgcagat gtcgacctca acttgagcat atctcaacca 2520

acttcacaaa gtcccaaaag ggataagagc agccttggcc tgcaactcca ccatggatca 2580

tatgaaggct ctgaactaaa gagaccaaag gcaagtacaa atgctttctt taaaagatta 2640

ggcaaccagt ccaatttcac tgctggattt gtatggctac ctgatccact atgtgattaa 2700

taattgagtt tcactatcac catgctagtg actgcagtat gatgtcctat cgtaacaaag 2760

tctttttcct agtttacttc ataggaactt ctacagctat tgtcaaaatt aaatttcact 2820

gtcggctgac ccaccgccca aattatattc accacacagt aacatgttta cctttcctgc 2880

ctgactatgg ttatctatct atttttcatt cagaaattaa atagcttgag aagattatta 2940

gttgacattc aactggaaaa aatcttttgc atgttatgca ggtcgatgct ccccctgaga 3000

tggtcgcaat ccctcatcga tacccccttc tgaccgagca tccaccaatc tggcatggcc 3060

aatcatatcc ccatcttttt aaataatgag gtgaggtgat actaaatttt aaagctcctt 3120

tcacctaaaa ttttcaaggt ggccctgatg acgcccgata tggatcggac cgtacatatt 3180

gagcatgtcg acgccactct gttttgcaat caggactgac tgatgcagac tgatagtgcc 3240

cttgcagctg ttactgttgt acttagcgtc acttaagcat tttcctgtgt ggctgcagtt 3300

atagctcaag ttggcctgat tttcatagtc cacccgttcc atgtcgagtt cagagtcatt 3360

aattcctagc acaattttca tatgcaggat cttgagtcct cacaaactgt ctaattgagt 3420

agcactactc agagtcgtag gatatgtata gtataaatat gctgattttt ttcctcctaa 3480

aggaacatcc tagagagttg acgatttaga tttagcccac tccaataatt tggtcaatat 3540

tccttcatgt ggctaagaac gttttggtgt tctattttgt atggtatgac cactattcag 3600

aggttttatt tatttatagc gagaaaagag atctggatcg taccaatact attttaatag 3660

tatgagtacg gctcacagct ttacttggag gcaaaagaaa gctagcaagt cggcacctta 3720

tagtaatagg attagtacgc agtttgccat ttggtttgaa aacccaaaat tggtttgcca 3780

ttccttttct taaattatcc tctcagattc ctcaactact aaacttcaag agtatcttca 3840

catgtgtcat tttcttattt tatttttcct ggaaagcaag ttcagctgga catatctacc 3900

tcacctttat gtcgatcaaa gcgataggaa ttagaccaat tctacaaaag aaacaaagaa 3960

caatccaatt tctgccaatt ttcttgagtt tatcctacat gagcatgcta aattattctt 4020

catttatata tgcaggaagc agccagagat catagcagga ggccagaggt ggccacaggg 4080

gctgttccaa cctgggcatg gagggtgagc caccctcctc caacacaacc catgccactc 4140

ttctcgtcgt catcgtccgc tgcagcatca tcaggattct ccaaaaccgc cgcggcagct 4200

gcccccggcg ccccatcggc ctcattccgg ttcgacccga tggcgccatc atcatcgtca 4260

agcaaccaac accaccacca ccccccgctg aaaaagaagc catactgtaa attttctggg 4320

aagccagcat ctttttgccc ctccggcgtt tcagcgtttt cggtcttgcg ccggggcggt 4380

ttcatgtagt ggattggatt catgacttta tttcccatgc tgcccaagtg aaatgcccct 4440

tccatttttg cgctctctgc atcagcgcat tggtcccata attctcgatg tcagatacta 4500

acctcagctc atctcaccat ctgagatgga tttataccat tgttgtagac aaacctgtca 4560

ctgaaattca gcagtaccga taccataaga taagaggaac ctttactttg ggaattattc 4620

cagtcccttt tcttggatgt cagttggctc ttgtttttgc tctatagcca actaccactg 4680

ctgaattgta ctgttgtttg ttgttgcact gttacaagat ttggctctaa cggatgccaa 4740

agtatctggt gcaagaatat aaa 4763

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence (Artificial sequence)

<400> 3

tcttcttgaa tcgtgaggtt 20

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence (Artificial sequence)

<400> 4

gctgaggtta gtatctgaca 20

<210> 5

<211> 18

<212> DNA

<213>Artificial sequence (Artificial sequence)

<400> 5

tgaagttgct gctgaagg 18

<210> 6

<211> 18

<212> DNA

<213>Artificial sequence (Artificial sequence)

<400> 6

cgatgacgac gagaagag 18

<210> 7

<211> 527

<212> PRT

<213> WSR1（Wheat Starch Regulatory 1）

<400> 7

Met Glu Leu Asp Leu Asn Val Glu Glu Lys Pro Ala Ala Val Ala Arg

1 5 10 15

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

20 25 30

Cys Gly Gly Gly Ala Ala Pro Ala Glu Glu Ala Ser Ser Ser Thr Arg

35 40 45

Gln Pro Ala Pro Ala Pro Arg Ala Val Leu Glu Phe Ser Ile Leu Arg

50 55 60

Ser Ser Ala Ser Ala Glu Gly Glu Asn Asp Val Gly Ala Asp Asp Asp

65 70 75 80

Glu Glu Glu Ala Thr Pro Ser Pro Pro Pro Pro Pro Pro Arg His Tyr

85 90 95

His Gln His Leu Leu Gln Pro Gln Gln Leu Val Thr Gln Glu Leu Phe

100 105 110

Pro Ala Ala Ala Ala Gly Gly Gly Pro Pro Pro Met Pro Val Pro Gln

115 120 125

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

130 135 140

Met Arg Ile Leu Gln Leu Gln Gln Gln Ala His Ala Pro Pro Pro Pro

145 150 155 160

Pro Pro Ala Ala Gln Pro Pro Val Ala Lys Lys Ser Arg Arg Gly Pro

165 170 175

Arg Ser Arg Ser Ser Gln Tyr Arg Gly Val Thr Phe Tyr Arg Arg Thr

180 185 190

Gly Arg Trp Glu Ser His Ile Trp Asp Cys Gly Lys Gln Val Tyr Leu

195 200 205

Gly Gly Phe Asp Thr Ala His Ala Ala Ala Arg Ala Tyr Asp Arg Ala

210 215 220

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

225 230 235 240

Asp Tyr Glu Asp Asp Met Lys Gln Met Lys Gly Leu Ser Lys Glu Glu

245 250 255

Phe Val His Val Leu Arg Arg Gln Ser Thr Gly Phe Ser Arg Gly Ser

260 265 270

Ser Lys Tyr Arg Gly Val Thr Leu His Lys Cys Gly Arg Trp Glu Ala

275 280 285

Arg Met Gly Gln Phe Leu Gly Lys Lys Ala Tyr Asp Lys Ala Ala Ile

290 295 300

Lys Cys Asn Gly Arg Glu Ala Val Thr Asn Phe Glu Pro Ser Thr Tyr

305 310 315 320

Asp Ala Glu Leu Leu Asn Glu Val Ala Ala Glu Gly Ala Asp Val Asp

325 330 335

Leu Asn Leu Ser Ile Ser Gln Pro Thr Ser Gln Ser Pro Lys Arg Asp

340 345 350

Lys Ser Ser Leu Gly Leu Gln Leu His His Gly Ser Tyr Glu Gly Ser

355 360 365

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

370 375 380

Pro His Arg Tyr Pro Leu Leu Thr Glu His Pro Pro Ile Trp His Gly

385 390 395 400

Gln Ser Tyr Pro Leu Phe Leu Asn Asn Glu Glu Ala Ala Arg Asp His

405 410 415

Ser Arg Arg Pro Glu Val Ala Thr Gly Ala Val Pro Thr Trp Ala Trp

420 425 430

Arg Val Ser His Pro Pro Pro Thr Gln Pro Met Pro Leu Phe Ser Ser

435 440 445

Ser Ser Ser Ala Ala Ala Ser Ser Gly Phe Ser Lys Thr Ala Ala Ala

450 455 460

Ala Ala Pro Gly Ala Pro Ser Ala Ser Phe Arg Phe Asp Pro Met Ala

465 470 475 480

Pro Ser Ser Ser Ser Ser Asn Gln His His His His Pro Pro Leu Lys

485 490 495

Lys Lys Pro Tyr Cys Lys Phe Ser Gly Lys Pro Ala Ser Phe Cys Pro

500 505 510

Ser Gly Val Ser Ala Phe Ser Val Leu Arg Arg Gly Gly Phe Met

515 520 525

Claims

1. a kind of transcription factor WSR1 genes inhibiting wheaten starch synthesis, which is characterized in that including a) or b) any described Nucleotide sequence：

A) nucleotide sequence shown in SEQ ID NO.1；

B) nucleotide sequence shown in SEQ ID NO.1 is through replacing, the core with same function of missing or addition base formation Nucleotide sequence.

2. a kind of albumen of the transcription factor WSR1 gene codes described in claim 1 for inhibiting wheaten starch to synthesize.

3. albumen according to claim 2, which is characterized in that its amino acid is as shown in SEQ ID NO.7.

4. a kind of transcription factor WSR1 gene tables for inhibiting wheaten starch synthesis containing nucleotide sequence described in claim 1 Up to carrier.

5. a kind of host cell containing the expression vector described in claim 4.

6. a kind of transcription factor WSR1 genes described in claim 1 inhibiting wheaten starch synthesis are cultivating high starch transgenosis Application in plant.

7. application according to claim 6, which is characterized in that the high starch genetically modified plants are wheat.

8. a kind of breeding method of the transgenic wheat with high starch, which is characterized in that be overexpressed SEQ ID in wheat NO：Gene shown in 1 inhibits SEQ ID NO：The expression of gene shown in 1, to cultivate the transgenic wheat with high starch.

9. a kind of transcription factor WSR1 genes described in claim 1 inhibiting wheaten starch synthesis are cultivating high yield transgenosis Application in plant.

10. application according to claim 9, which is characterized in that the high yield genetically modified plants are wheat.