CN106317211A

CN106317211A - Rape grain weight related gene ARF 18 and application thereof

Info

Publication number: CN106317211A
Application number: CN201510381235.4A
Authority: CN
Inventors: 王汉中; 华玮; 刘静; 胡志勇; 杨红丽; 张亮; 王新发
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2015-07-02
Filing date: 2015-07-02
Publication date: 2017-01-11

Abstract

The invention provides a rape grain weight related gene ARF 18 and an application thereof. The rape grain weight can be controlled by virtue of the ARF18 gene by map-based cloning, and the ARF gene sequence is shown as SEQ ID NO.2. The ARF gene has great promotion effect on the molecular mechanism research on plant grain weight regulation and control. The cloned rape ARF18 gene has an obvious effect of regulating and controlling the grain weight of rape, this is of great significance in clarifying the biological function of the ARF18 gene. By adopting the gene engineering technology, the expression quantity of the ARF18 gene can be increased or weakened, so as to regulate and control the rape seed, so that the balance between the three factors of the yield is facilitated, and therefore the purpose of increasing the yield is achieved.

Description

One and rape grain heavy phase correlation gene ARF18 and application thereof

Technical field

The invention belongs to plant genetic engineering field.Specifically, the present invention relates to a kind of and rape grain heavy phase correlation gene ARF18 and application thereof.

Background technology

Along with the quick growth of population, global food shortage problem becomes increasingly severe.Therefore, it is badly in need of breaking through in terms of crop yield.As one of three factors affecting crop yield, grain weight has been widely regarded as the complicated quantitative trait of controlled by multiple genes.Extremely important for improving crop breeding efficiency to the heredity of grain principal characteristic shape and the parsing of molecular mechanism.

Seed size is affected by a series of cell processes.This impact derives from parents' effect and parent/zygote tissue (Fang et al., 2012).In model plant arabidopsis, some mutants such as ap2, arf2, da1, eod3, ttg2, klu etc. mainly extended by the cell of regulation and control seed coat control seed size (Fang et al., 2012；Ohto et al.,2005；Schruff et al.,2006；Garcia et al.,2005；Adamski et al., 2009), and in mini3, iku1, iku2 and shb1 mutant, endosperm seed development premature cell or increment in early days have impact on seed size (Garcia et al., 2003；Luo et al.,2005；Wang et al.,2010；Zhou et al.,2009；Kang et al., 2013) .met1 gene due in CG background the methylated disappearance of cytosine residues cause that seed size is had parents' effect (Feng et al., 2010).In Oryza sativa L., identified 47 seed length QTLs and 48 Kernel-Width QTLs (Bao, 2014), some regulation and control seed sizes gene such as GW2, GIF1, qSW5, GS3, GS5, GW8 and qGL3 be positioned (Song et al., 2007；Wang et al.,2008；Shomura et al.,2008；Weng et al.,2008；Mao et al.,2010；Li et al.,2011；Wang et al.,2012；Zhang et al.,2012).Wherein, GW2 and qSW5 affects grain weight by regulating coetonium hülle cell number, and other gene is then directly to regulate seed cell number or cell size to affect grain weight.Although grain heavily research obtains good progress, at present still without the new gene finding regulation and control grain weight in polyploid Brassica campestris L.

Polyploid is by monoploid polyploidization or the combination of two or more diversity gene groups, and the latter generally exists (Masterson, 1994) in flowering plant includes many important crops.Polyploid genome due on coloured differently body the existence of homologous sequence and homogenic interaction make QTL location more difficult.Such as cabbage type rape (AACC), the in the world second largest oil crop after Semen sojae atricolor, it is to be planted natural hybridization by Chinese cabbage and two ancestors of Caulis et Folium Brassicae capitatae.Chinese cabbage is A genome.A genome and the C genome similarity the highest (Rana et al., 2004) of Caulis et Folium Brassicae capitatae.Recent years, as the agricultural crops that the whole world is important, Brassica campestris L receives more and more attention.So far, the QTL closed despite more than 80 grain heavy phases is identified out, there is not been reported for concrete controlling gene (Quijada et al., 2006；Udall et al.,2006；Fan et al.,2010；Yang et al.,2012).

In the research of our early stage, the F2 segregating population utilizing zy72360 and R1 to build detects a grain weight main effect QTL being positioned on A9 chromosome, and its contribution rate is more than 30%.In the present invention, we are by building the grain weight controlling gene in NIL and sources group association analysis map based cloning A9 site.Wherein, ARF18 is as the candidate gene of regulation and control grain weight, and Land use models plant Arabidopsis thaliana and Brassica campestris L have made further functional analysis.

Summary of the invention

It is an object of the invention to there are provided a kind of and rape grain heavy phase correlation gene ARF18, its sequence is shown in SEQ ID NO:2.The protein of coding is shown in SEQ ID NO.3.

Another object of the present invention is to there are provided a kind of application with rape grain heavy phase correlation gene ARF18 in improving plant grain principal characteristic shape.

In order to realize foregoing invention purpose, the present invention uses techniques below measure:

One and rape grain heavy phase correlation gene ARF18, obtain in the following manner:

1. the NIL in a weight main effect QTL site builds

Parent zy72360 and R1 has significant difference in terms of grain principal characteristic shape.Carry out just positioning to grain principal characteristic shape QTL by F2 colony.Utilize Hybrids F1 and parent's continuous backcross 4 times, period utilize QTL linked marker combine phenotype carry out heredity prospect selection, utilize non-linked marker to carry out the assessment of genetic background simultaneously, from BC4F1 colony, obtain target QTL heterozygosis and the high QTL-NIL individual plant of genetic background response rate；BC4F1 is utilized to obtain BC4F2 for QTL-NIL segregating population for heterozygosis target QTL-NIL individual plant selfing.

2. the fine location in a weight main effect QTL site

Based on the primer that this interval is newly developed, we utilize NIL BC4F2 colony's shortening interval.According to the SNP information in interval, continue to shorten interval by PCR amplification. the distance interval in order to shorten QTL further, utilize 380 parts of sources group, develop and screen the labeled primer obtained in interval and carry out the association analysis between grain weight phenotype and genotype.According to linkage analysis and association analysis, we the most at last this deciding field to finding 7 genes within 50kb and in this region.

3. determine mutational site, it is thus achieved that candidate gene

According to rapeseed gene group sequence, we utilize zy72360 and R1 material to clone coding region and the upstream regulatory region of above-mentioned 7 genes, and alignment finds that wherein 4 genes exist SNP and In/Del.Wherein the variation of ARF18 gene is maximum, is classified as the candidate gene in regulation and control grain weight main effect QTL site.

The functional verification of 4.ARF18 gene

Land use models plant Arabidopsis thaliana is receptor, and overexpression ARF18 and allele thereof verify its function.Building overexpression vector, use the genetic transforming method of Agrobacterium EHA105 mediation, imported by overexpression vector in arabidopsis, last each gene obtains transgenic line about 30 strain of overexpression.Obtain the homozygous lines of transgenic line, investigate grain weight phenotype, finally found that the overexpression of ARF18 (shown in SEQ ID NO.2) can reduce seed weight.By the investigation of the arabidopsis homologous genes mutation type surface to this gene, find that the seed grain of mutant material heavily increases.This shows that the expression height of ARF18 can regulate and control the weight of plant seed.

Being finally obtained a kind of and rape grain heavy phase correlation gene ARF18, its sequence is shown in SEQ ID NO.2.

A kind of application with rape grain heavy phase correlation gene ARF18 in improving rape grain principal characteristic shape, including by biotechnology, suppresses the expression of ARF18 gene in Brassica campestris L, it is thus achieved that the seed grain of transgenic plant heavily increase.

The protection content of the present invention also includes; with the application in improving plant grain principal characteristic shape of the phytoprotein of protein homology shown in SEQ ID NO.3; plant will be suppressed in expression with the phytoprotein of protein homology shown in SEQ ID NO.3, it is thus achieved that the seed grain of transgenic plant heavily increase.

Term " transgenic plant " used in the present invention refers to the gene containing importing and gene expression that stably enhancer or inhibitor is imported generation can have the plant of specific biological character.The plant being previously mentioned in the present invention includes the cereal crops such as Oryza sativa L., Semen Tritici aestivi, Semen Maydis, also includes the industrial crops such as Brassica campestris L, Semen sojae atricolor, Cotton Gossypii, also includes the vegetable crops such as the Fructus Cucumidis sativi, the Fructus Lycopersici esculenti that grow summer, also includes arabidopsis.

Compared with prior art, the invention have the advantages that

Not yet navigating to grain readjustment control related gene at present in Brassica campestris L, the present invention can control rape grain weight by the ARF18 gene of map based cloning, and this molecular mechanism research that plant grain is resetted control will have great impetus.Although the gene order of ARF18 is disclosed in arabidopsis, but and do not know its concrete biological function, the Brassica campestris L ARF18 gene pairs rape grain of present invention clone heavily has significant regulating and controlling effect, and this is significant to the biological function illustrating ARF18 gene.The expression being improved or weakening ARF18 gene by technique for gene engineering can regulate and control the size of Semen Brassicae campestris, thus beneficially balance between yield three factor and then reach the purpose of volume increase.

Accompanying drawing explanation

The grain weight phenotype analytical of Fig. 1 two parent zy72360 and R1.

Grain weight QTL interval is foreshortened to 120kb by Fig. 2 NIL linkage analysis.

Fig. 3 sources group association analysis finds this QTL segment part labelling and grain principal characteristic shape pole significant correlation.

In Fig. 4 zy72360 and R1 material, the sequence difference of ARF18 gene compares.

Fig. 5 reduces seed weight from the overexpression in arabidopsis of the ARF18 in R1 material, and significantly changes from the gene overexpression in zy72360 without result in seed.

Fig. 6 arabidopsis ARF18 mutant seeds weight increases.

The expression vector of Fig. 7 ARF18RNAi converts R1 and causes the seed grain of transgenic line heavily to increase.

Detailed description of the invention

Generally according to normal condition such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or Draper et al. (Blackwell Science Press, 1988) condition described in, or according to the condition proposed by agents useful for same manufacturer.

Embodiment 1:

The NIL in grain weight main effect QTL site builds

Parent cabbage type rape zy72360 and R1 (Hu Z, Hua W, Huang S, Yang H, Zhan G, Wang X, et al. (2012) Discovery of Pod Shatter-Resistant Associated SNPs by Deep Sequen cing of a Representative Library Followed by Bulk Segregant Analysis in Rapesee D.PLoS ONE 7 (4): e34253.) there is in terms of grain principal characteristic shape significant difference (zy72360 be 5.53 ± 0.43g, R1 are 3.98 ± 0.51g) (Fig. 1).First, SW QTL is navigated between W236 and W239 by we by F2 colony.Utilize Hybrids F1 and parent's continuous backcross 4 times, period utilize QTL linked marker combine phenotype carry out heredity prospect selection, utilize non-linked marker to carry out the assessment of genetic background simultaneously, from BC4F1 colony, obtain target QTL heterozygosis and the high QTL-NIL individual plant of genetic background response rate；BC4F1 is utilized to obtain BC4F2 for QTL-NIL segregating population for heterozygosis target QTL-NIL individual plant selfing.This colony of field planting, 3800 individual plants of final acquisition.

Embodiment 2: the fine location in grain weight main effect QTL site

Based on the primer that this interval is newly developed, we utilize NIL BC4F2 colony, and this interval is foreshortened to the 147kb between W024 and W004.Utilize the SNP information within 147kb, continue to foreshorten to 120kb (Fig. 2) by interval by PCR amplification. the distance interval in order to shorten QTL further, utilize 380 parts of sources group, develop and screen the labeled primer obtained in 11 QTL intervals and carry out the association analysis between grain weight phenotype and genotype.Result shows that the labelling between SSR-72and SSR-89 shows significant relatedness (Fig. 3).According to linkage analysis and association analysis, we the most at last this deciding field within 50kb.

Embodiment 3:

Determine mutational site, it is thus achieved that candidate gene

According to rapeseed gene group sequence, design the both sides primer across upstream region of gene control region and gene regions and the gene coding region primer of amplifiable cDNA library, for expanding the corresponding sequence of candidate gene from Brassica campestris L.

3.1 rapeseed gene groups DNA and the extraction of total serum IgE

The extraction of DNA:

A.70% blade cleaned by (volume ratio) ethanol, weighs about 100mg

B. adding 600ul extraction buffer (0.2M Tris-Cl, 0.25NaCl, 25mM EDTA, 0.5%SDS, pH 7.5), room temperature is quickly ground.

C.1.5ml Ependorff pipe mesoscale eddies mixing 5-10s.

D.12000rpm, 25min, room temperature.Take supernatant, add equal-volume isopropanol ,-20 degrees Celsius of precipitates overnight.

E.12000rpm, 15min, room temperature.Add 70% ethanol 200ul foam washing DNA precipitation.

F.12000rpm, 15min, room temperature.Remove ethanol.It is inverted on napkin, treats that ethanol volatilization is clean.

G. add sterilized water 100ul dissolving and slightly put forward DNA precipitation.Its concentration is estimated with spectrophotometric determination or electrophoresis.

The extraction (TRIZOL TM Kit extracts RNA) of RNA

Liquid nitrogen grinding 100mg material

A. adding 1mlTRIZOL, room temperature (20-25 DEG C, lower same) places 5min.

B. adding 200ul chloroform, acutely vibrate 30s, and room temperature places 2min.

C.12000g, 15min, 4 DEG C, take supernatant in new pipe, add 500ul isopropanol, after mixing, room temperature places 15min.

D.12000g, 15min, 4 DEG C, removes supernatant, adds 1ml70% (anhydrous alcohol and the volume ratio of H2O) ethanol.

E.7500g, 7min, goes supernatant, air to be dried by 4 DEG C.

F.DEPC-H₂O dissolves.

3.2 the amplification of genome sequence

With the genomic DNA of zy72360 and R1 as template, the upstream promoter control region of 7 genes of amplification and genome sequence.PCR response time and temperature are made as follows: 94 DEG C of 3min, 94 DEG C of 45s, 59 DEG C of 45s, 72 DEG C of 2min 30s, 30cycles, 72 DEG C 5min.

The amplification of 3.3 gene coding regions

For the amplification of coding region, first, the total serum IgE utilizing zy72360 and R1 carries out reverse transcription.The reverse transcription of cDNA the first chain uses RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas), operates and carries out with reference to used kit explanation.Carry out PCR amplification, PCR response time and temperature with cDNA for template and make as follows: 94 DEG C of 3min, 94 DEG C of 45s, 59 DEG C of 45s, 72 DEG C of 2min 30s, 30cycles, 72 DEG C 5min.

The extension increasing sequence of 3.4 above-mentioned acquisitions is compared between two parents, finds that the coding region of ARF18 exists larger difference (lost an exon in zy72360) (Fig. 4), is therefore classified as candidate gene and carries out transgenic functional verification.

Embodiment 4: the functional verification of candidate gene

The structure of 4.1 expression vectors and the conversion of arabidopsis

After the coding region sequence of ARF18 (shown in SEQ ID NO.2) and allele (shown in SEQ ID NO.1) thereof is connected with TOPO entry vector (invitrogen company), it is transformed in competent cell DH5 α (invitrogen company), grand enzyme element screens, with gene primer (upstream region of gene primer) amplification, vector primer (T7 primer) identifies that forward inserts clone, plasmid is recombinated with Pearleygate100 (invitrogen company) after preparation in a small amount, and be transformed in competent cell DH5 α, kanamycin screens, its Insert Fragment is identified with gene primer (downstream of gene primer) PCR through vector primer (35S starts word aligning primer).

The conversion process of arabidopsis:

Preparation of reagents

Osmotic medium (1L): 1/2xMurashige-Skoog；5% (mass ratio) sucrose；0.5 gram of MES；It is adjusted to pH5.7 with KOH；Add again: the 6-BA mother solution of 10 microlitre 1mg/ml；200 microlitre Silwet L-77

Step of converting

(1) prepare Agrobacterium (Agrobacterium tumefaciems GV3101) bacterium solution 10ml having converted corresponding plasmid, converting evening before that day, proceed to big bottle overnight incubation, within second day, take out agrobacterium liquid O.D600 when using and work as between 1.2 to 1.6.

(2) room temperature 5000rpm is centrifuged 15 minutes.

(3) abandon supernatant, Agrobacterium precipitation is suspended in the osmotic medium of respective volume, makes O.D600 about 0.8.

(4) whole plant is directly dipped to agrobacterium suspension 30s.

(5) lucifuge overnight incubation, then normally cultivates to knot.

The screening of 4.2 transgenic arabidopsis and checking

The screening of transformant

By the arabidopsis seed of vernalization in the Artificial Soil watering supersaturation PNS nutritional solution, and with on preservative film cover.Manual simulation natural conditions illumination two days later (illumination 16 hours, dark 8 hours), took off film after three days.

Artificial culture room condition: relative humidity 80%, constant temperature 20-240C, intensity of illumination 80-200umol/M2/S, periodicity of illumination is 8h dark 16h illumination cultivation.About one week, spray herbicide screening positive plant.

PCR identifies

(1) extraction of the transformed plant STb gene of PCR it is used for

A.70% blade cleaned by (volume ratio) ethanol, weighs about 100mg

C.1.5ml Ependorff pipe mesoscale eddies mixing 5-10s.

H. with STb gene as template, PCR is carried out.

(2) PCR program

PCR reaction utilizes 35S promoter sequence and downstream of gene primer in plant expression vector [5 '-TCAGCCAGAGATCTGGATAG-3 '], and the time of reaction and temperature are made as follows:

94℃3min

94 DEG C of 45s,

59℃45s

72 DEG C of 2min 30s, 30cycles

72℃5min

Testing result shows, most of transformed plants all can amplify the electrophoretic band of expection size, and negative control does not then have, and shows to have contained in transgenic arabidopsis genome exogenous gene DNA fragmentation.

4.3 the phenotype of transgenic line is investigated

Homozygous lines to transgenic line carries out the investigation of grain weight phenotype, find that overexpression reduces the seed weight of partial transgenic strain from the ARF18 in R1 material, reduction amplitude is at about 10% (Fig. 5), overexpression is then the most consistent with wild type control from the ARF18 in zy72360 material, and seed weight does not has significant change.

By the investigation of arabidopsis homologous genes deletion mutation strain (commercial source: https: the //www.arabidopsis.org/) phenotype to this gene, find that the seed grain of mutant material heavily increases (Fig. 6).This shows that the expression height of ARF18 can regulate and control the weight of plant seed.

Embodiment 5:

The structure of Brassica campestris L ARF18 suppression expression vector and the conversion in Brassica campestris L

The vector construction that 5.1CaMV promoters driven ARF18 antisense RNA is expressed

ARF18 coding region sequence according to R1 material, design primer amplification partial coding region fragment (300bp) primer sequence is: ARF18RNAiF:5 '-atggcgaatgtagatggagatga-3 ', ARF18RNAiR:5 '-tgaagtctacgctcagatcacat/atgtgatctgagcgtagacttca-3 '.It is reversely connected on topo carrier and is recombinated in pEarleyGate100.Brassica campestris L is converted by vector to LBA4404 prepares.

5.2 the Agrobacterium LBA4404 of Brassica campestris L converts

A. the preparation of aseptic seedling: seed, through 70% ethanol 1min, after mercuric chloride (HgCl2) immersion 13-15min, ddH2O wash 5 times, is laid in MS culture medium (PH 5.8), agar concentration 0.8%.Brassica campestris L aseptic seedling is standby.

B. coleseed petiole converts: inoculation agrobacterium tumefaciens lba4404 is on solid medium LB, and picking list bacterium colony is cultivated in 50ml YEP fluid medium (tryptone 10g+ yeast extract 10g+NaCl5g+MgSO40.5g) two days later.Take 4-5 days aseptic seedling cotyledon petioles, in bacterium solution, soak 5-8min, therebetween jog, then go bacterium solution, suck residual bacterium solution in outer implant with aseptic filter paper.It is placed in and co-cultures base (MS+0.2mg/L 6-benzyladenine (6-BA)+1mg/L 2,4-dichlorphenoxyacetic acid (2,4-D)+200um acetosyringone (AS), PH5.8), upper cultivate 2-3 days.

C. the outer implant after co-culturing proceeds to containing only in the penicillin (Car) division culture medium without Kan, carries out de-bacterium and differentiation culture 5-7 days in the greenhouse of dark condition.Followed by generation at Selective agar medium (MS+3mg/L6-BA+0.1mg/L α-naphthaleneacetic acid (NAA)+5mg/L silver nitrate (the AgNO3)+400mg/L Car+15mg/L Kan adding Kan screening pressure；PH5.8) in, screen, regenerated green bud to be differentiated.

Cut budlet when bud length the most to be regenerated is to 1cm and move to root media (MS+0.2mg/L NAA+10mg/L Kan+400mg/L Car；PH5.8) on, with the solidified MS media screening adding Car Yu Kan.

E., after seedling rooting, it is transplanted into outdoor.Lower soil alms bowl is transplanted after 10 days.

Embodiment 6: the qualification of transformed plant

(1) extraction of the transformed plant STb gene of PCR it is used for

A.70% blade cleaned by ethanol, weighs about 100mg

C.1.5ml Ependorff pipe mesoscale eddies mixing 5-10s.

H. with STb gene as template, PCR is carried out.

(2) PCR program

The cumulative volume of PCR reaction system is 20 μ l, genomic DNA template 1ul (about 50ng), 1 × Taq enzyme reaction buffer, 25mM MgCL2 1.2ul, each 0.2ul of 2mM dNTP 1.5ul, 10uM primer, 50% glycerol 2ul, 0.3 unit rTaq enzyme (Takara company), add ddH2O to 20 μ l.The PCR primer across intron is designed and synthesized according to genes of interest in plant expression vector.(forward primer: [5 '-CACGTCTTCAAAGCAAGTGGA-3 '] and reverse primer [5 '-atcagaaatatggtcagtcac-3 '], the time of reaction and temperature are made as follows: 94 DEG C of 3min, 94 DEG C of 45s, 59 DEG C of 45s, 72 DEG C of 2min 30s, 30cycles, 72 DEG C 5min.

Testing result shows, positive control and most of transformed plant all can amplify the electrophoretic band of expection size, and negative control does not then have, and shows to have contained in transgene rape genome exogenous gene DNA fragmentation.

Embodiment 7: T1 is carried out phenotype analytical for transgenic line

Gathering in the crops after Brassica campestris L ARF18RNAi transgenic line and comparison full maturity, Semen Brassicae campestris threshing is also fully dried and is weighed mass of 1000 kernel.Final result shows, compared with non-transgenic strain, partial transgenic strain seed weight increases, and maximum amplification is more than 15% (Fig. 7).

SEQUENCE LISTING

<110>Inst. of Oil Crops, Chinese Academy of Agriculture

<120>one and rape grain heavy phase correlation gene ARF18 and application thereof

<130>one and rape grain heavy phase correlation gene ARF18 and application thereof

<160> 3

<170> PatentIn version 3.1

<210> 1

<211> 1521

<212> DNA

<213>artificial sequence

<400> 1

atggcgaatg tagatggaga tgattccaga agttctttcc caagttgtta tcaggatcag 60

ctgtacacag agctatggaa agcctgtgca ggtccattag tggaggttcc tcttgttgga 120

gagagagttt tctacttccc tcagggtcac atggaacaac ttgtggcctc aactaatcaa 180

ggaattgaat cagagaaaat acctgatttt aaacttcctc ccaagatact ctgtcaagtt 240

cttagtgtga tgctaaaggc agagcatgac acagatgaag tctacgctca gatcacatta 300

aaaccagagg aagatcaaag tgaaccaaca agtcttgatc caccaattgt tgaaccaaca 360

aagcaaatgt tccactcttt tgtaaagatt ctaacagctt cagacacaag cactcatgga 420

gggttctctg ttcttcgtaa acacgccact gaatgcttgc ctgccttgga catgacacaa 480

gctactccta ctcaagaact tgtgactaga gatcttcatg ggtttgagtg gaggtttaag 540

catattttca gaggacaacc taggaggcat ttgcttacta caggctggag tacctttgtt 600

tcctcaaaaa gacttgtagc tggagatgct tttgtgttct tgaggataag ccaattcata 660

gtaggtgtga acaagtatat ggaagctatg aagcatggtt tctctcttgg tacaagattc 720

aggatgaagt ttgaaggaga agagtctcct gagagaatat ttaccggtac tattgtggga 780

attggagatt tatcttcaca atggccagct tctacatgga gatcattgca ggtccaatgg 840

gatgagccaa caacagttca gagaccagac aaagtctcac catgggagat tgagcctttc 900

ttgccatctt ccccagcttc aacaccttct caacaatcac aacccaaaag caaaaggtca 960

aaacctgttg aatcatcaag tttgagtcca ggtcaagcta gtttcttagg cgtccaagct 1020

gagcctcctc ctcctcctcc tcctgcgagt agttgctata ggttgtttgg atttgatctc 1080

acaagcaatc ctccagctcc aatacctcca gacaagcaac cgatggatac ttctgaagct 1140

gccaagtgtc aagaccccat cactccaagc tcagttaatg agccaaagaa gcaacaaaca 1200

tcaaggactc gaaccaaagt gcaaatgcaa gggatagctg ttggtcgtgc ggtagattta 1260

acgctgttga aatcatatga tgaactgatt aaggagcttg aggagatgtt tgagatccaa 1320

ggacagcttc ttccccgaga taaatggatc gttgtcttca ctgatgatga aggtgacatg 1380

atgcttgctg gagatgatcc atggaatgag ttttgtaaga tggcgaagaa gatatttata 1440

tattcaagcg atgaggttaa gaaaatgaca aggagaatga agagttcttc ttcgttagag 1500

aatgaagcaa gcatggatta a 1521

<210> 2

<211> 1680

<212> DNA

<213>artificial sequence

<400> 2

atggcgaatg tagatggaga tgattccaga agttctttcc caagttgtta tcaggatcag 60

ctgtacacag agctatggaa agcctgtgca ggtccattag tggaggttcc tcttgttgga 120

gaaagagttt tctacttccc tcagggtcac atggaacaac ttgtggcctc aactaatcaa 180

ggaattgaat cagagaaaat acctgatttt aaacttcctc ccaagatact ctgtcaagtt 240

cttagtgtga tgttaaaggc agagcatgac acagatgaag tctacgctca gatcacatta 300

aaaccagagg aagatcaaag tgaacctaca agtcttgatc caccaattgt tgaaccaaca 360

aagcaaatgt tccactcctt tgtaaagatt ctaaccgctt cagacacaag cactcatggt 420

ggattctctg ttcttcgtaa acacgccact gaatgcttgc ctgccttgga catgacacaa 480

gctattccta ctcaagaact tgtgactaga gatcttcatg ggtttgagtg gaggtttaag 540

catattttca gaggacaacc taggaggcat ttgcttacta caggctggag tacatttgtt 600

tcctcaaaaa gacttgtagc tggagatgct tttgtgttct tgaggggtga gaatggagat 660

ttaagagttg gagtgaggcg tttagctagg catcagaaca ccatgcctgc ttcagttatc 720

tctagtcaga gcatgcattt aggagtcctt gctacagctt ctcatgctgt gaacacccaa 780

actatgtttc ttgtgcttta caagcctagg ataagccaat tcatagtaag tgtgaacaag 840

tatatggaag ctatgaagca tggtttctct cttggtacaa gattcaggat gaggtttgaa 900

ggagaagagt ctcctgagag aatatttacc ggtactattg tgggaattgg agatttatct 960

tcacaatggc cagcttctac atggagatca ttgcaggtcc aatgggatga gccaacaaca 1020

gttcagagac cagacaaagt ctcaccatgg gagattgagc ctttcttgcc atcttcccca 1080

gcttcaacac cttctcaaca atcacaaccc aaaagcaaaa ggtcaaaacc cattgaatca 1140

tcaagtttga gtccaggtca agctagtttc ttaggcgtcc aagctgagcc tcctcctcct 1200

cctgcgagta gttgctatag gttgtttgga tttgatctca caagcaatcc tccagctcca 1260

atacctccag acaagcaacc gatggatact tctgaagctg ccaagtgtca agaccccatc 1320

actccaagct cagttaatga gccaaagaag caacaaacat caaggactcg aaccaaagtg 1380

caaatgcaag ggatagctgt tggtcgtgcg gtagatttaa cgctgttgaa atcatatgat 1440

gaactgatta aggagcttga ggagatgttt gagatccaag gacagcttct tccccgagat 1500

aaatggatcg ttgtcttcac tgatgatgaa ggtgacatga tgcttgctgg agatgatcca 1560

tggaatgagt tttgtaagat ggcgaagaag atatttatat attcaagcga tgaggttaag 1620

aaaatgacaa ggagaatgaa gagttcttct tcgttagaga atgaagcaag catggattaa 1680

<210> 3

<211> 559

<212> PRT

<213>artificial sequence

<400> 3

Met Ala Asn Val Asp Gly Asp Asp Ser Arg Ser Ser Phe Pro Ser Cys

1 5 10 15

Tyr Gln Asp Gln Leu Tyr Thr Glu Leu Trp Lys Ala Cys Ala Gly Pro

20 25 30

Leu Val Glu Val Pro Leu Val Gly Glu Arg Val Phe Tyr Phe Pro Gln

35 40 45

Gly His Met Glu Gln Leu Val Ala Ser Thr Asn Gln Gly Ile Glu Ser

50 55 60

Glu Lys Ile Pro Asp Phe Lys Leu Pro Pro Lys Ile Leu Cys Gln Val

65 70 75 80

Leu Ser Val Met Leu Lys Ala Glu His Asp Thr Asp Glu Val Tyr Ala

85 90 95

Gln Ile Thr Leu Lys Pro Glu Glu Asp Gln Ser Glu Pro Thr Ser Leu

100 105 110

Asp Pro Pro Ile Val Glu Pro Thr Lys Gln Met Phe His Ser Phe Val

115 120 125

Lys Ile Leu Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe Ser Val

130 135 140

Leu Arg Lys His Ala Thr Glu Cys Leu Pro Ala Leu Asp Met Thr Gln

145 150 155 160

Ala Ile Pro Thr Gln Glu Leu Val Thr Arg Asp Leu His Gly Phe Glu

165 170 175

Trp Arg Phe Lys His Ile Phe Arg Gly Gln Pro Arg Arg His Leu Leu

180 185 190

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

195 200 205

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

210 215 220

Val Arg Arg Leu Ala Arg His Gln Asn Thr Met Pro Ala Ser Val Ile

225 230 235 240

Ser Ser Gln Ser Met His Leu Gly Val Leu Ala Thr Ala Ser His Ala

245 250 255

Val Asn Thr Gln Thr Met Phe Leu Val Leu Tyr Lys Pro Arg Ile Ser

260 265 270

Gln Phe Ile Val Ser Val Asn Lys Tyr Met Glu Ala Met Lys His Gly

275 280 285

Phe Ser Leu Gly Thr Arg Phe Arg Met Arg Phe Glu Gly Glu Glu Ser

290 295 300

Pro Glu Arg Ile Phe Thr Gly Thr Ile Val Gly Ile Gly Asp Leu Ser

305 310 315 320

Ser Gln Trp Pro Ala Ser Thr Trp Arg Ser Leu Gln Val Gln Trp Asp

325 330 335

Glu Pro Thr Thr Val Gln Arg Pro Asp Lys Val Ser Pro Trp Glu Ile

340 345 350

Glu Pro Phe Leu Pro Ser Ser Pro Ala Ser Thr Pro Ser Gln Gln Ser

355 360 365

Gln Pro Lys Ser Lys Arg Ser Lys Pro Ile Glu Ser Ser Ser Leu Ser

370 375 380

Pro Gly Gln Ala Ser Phe Leu Gly Val Gln Ala Glu Pro Pro Pro Pro

385 390 395 400

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

405 410 415

Pro Pro Ala Pro Ile Pro Pro Asp Lys Gln Pro Met Asp Thr Ser Glu

420 425 430

Ala Ala Lys Cys Gln Asp Pro Ile Thr Pro Ser Ser Val Asn Glu Pro

435 440 445

Lys Lys Gln Gln Thr Ser Arg Thr Arg Thr Lys Val Gln Met Gln Gly

450 455 460

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

465 470 475 480

Glu Leu Ile Lys Glu Leu Glu Glu Met Phe Glu Ile Gln Gly Gln Leu

485 490 495

Leu Pro Arg Asp Lys Trp Ile Val Val Phe Thr Asp Asp Glu Gly Asp

500 505 510

Met Met Leu Ala Gly Asp Asp Pro Trp Asn Glu Phe Cys Lys Met Ala

515 520 525

Lys Lys Ile Phe Ile Tyr Ser Ser Asp Glu Val Lys Lys Met Thr Arg

530 535 540

Arg Met Lys Ser Ser Ser Ser Leu Glu Asn Glu Ala Ser Met Asp

545 550 555

Claims

1. the protein separated, its sequence is shown in SEQ ID NO.3.

2. the nucleotide sequence of protein described in coding claim 1.

Nucleotide sequence the most according to claim 2, described sequence is shown in SEQ ID NO.2.

4. the protein described in claim 1 or the nucleotides sequence described in claim 2 are listed in the application improved in rape grain principal characteristic shape.

5. with the phytoprotein sequence of protein homology described in claim 1 in the application improved in plant principal characteristic shape.

Application the most according to claim 5, described plant is arabidopsis.