CN109136232A - Haynaldia villosa mildew-resistance gene DvRGA-1, DvRGA-2 and its application - Google Patents

Abstract

The invention discloses haynaldia villosa mildew-resistance gene DvRGA-1, DvRGA-2 and its applications, belong to genetic engineering field.The genomic dna sequence of haynaldia villosa DvRGA-1 is SEQ ID NO.1, and cDNA sequence is SEQ ID NO.2, and amino acid sequence is SEQ ID NO.3.The genomic dna sequence of DvRGA-2 is SEQ ID NO.4, and cDNA sequence is SEQ ID NO.5, and amino acid sequence is SEQ ID NO.6.DvRGA-1 is located at haynaldia villosa powdery mildew resistance gene Pm 21 site.Gene silencing analysis shows that, the silencing of DvRGA-1 and DvRGA-2 can lead to anti-disease wheat and sends out powdery mildew completely；Transient Expression analysis shows, DvRGA-1 and DvRGA-2 can significantly lower the powdery mildew haustorium index of susceptible wheat.Therefore, the protein of haynaldia villosa DvRGA-1 and DvRGA-2 gene and its coding has significant application value in wheat anti-powdery mildew breeding.

Description

Haynaldia villosa mildew-resistance gene DvRGA-1, DvRGA-2 and its application

Technical field

The invention discloses two haynaldia villosas (Dasyprum villosum) mildew-resistance gene DvRGA-1 and DvRGA- 2, they are all disease resistance gene analog (RGA), encode typical helix-coil-nucleotide binding site-rich in leucine (CC-NBS-LRR) class ill-resistant protein is repeated, wheat can be improved to the resistance of powdery mildew, belong to genetic engineering field.

Background technique

Wheat is one of world's staple food crop, produces the threat by various diseases.Wheat powdery mildew be have it is small Fungal disease caused by wheat powdery mildew (Blumeria graminis f.sp.tritici), be cause wheat yield it is important because Element.The economical and effective mode of control wheat powdery mildew harm is to excavate Resistant germplasm, cultivate disease-resistant variety.It has reported at present A powdery mildew resistance gene in wheat more than 100 (McIntosh et al.Catalogue of gene symbols for wheat: 2017supplement.In:KOMUGI wheat genetic resource database:https: // Shigen.nig.ac.jp/wheat/komugi/genes/symbolClass List.jsp), but microspecies spy is shown as mostly Resistance specificity, the gene with resistance of wide spectrum are less.Observation in recent years is shown, in In Middle And Lower Reaches of Changjiang River, powdery mildew microspecies Toxicity just gradually enhance, the isogenic wheat breed containing Pm2a, Pm4a planted extensively in the past just gradually loses resistance, to small Wheat production generates increasing threat (Bie et al.Development and characterization of an efficient breeding-practical marker MBH1simultaneously tagging Pm21and PmV genes conferring resistance to wheat powdery mildew.Molecular Breeding.2015, 35:189).The PmV gene of the Pm21 gene of haynaldia villosa from Britain Camb botanical garden and the haynaldia villosa from the former Soviet Union, all With resistance of wide spectrum, existing various wheat powdery mildew microspecies (Chen et al.Development and can be resisted molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew.Theor Appl Genet.1995,91:1125-1128；Li et al.Development and identification of wheat– Haynaldia villosa T6DL.6VS chromosome translocation lines conferring Resistance to powdery mildew.Plant Breed.2005,124:203-205), wherein being bred as more than 30 at present A wheat breed for carrying Pm21 gene, and the wheat breed for carrying PmV gene is relatively fewer.

Currently, as the isogenic substitution of Pm2a, Pm4a for increasingly losing resistance, Pm21 gene has obtained large area and has pushed away Extensively.But its mechanism of action is still not clear, this is mainly due to the translocation chromosomes and chromosome of wheat that carry Pm21 gene The limitation of the factors such as exchange does not occur, it is difficult to separate Pm21 using conventional map based cloning strategy.Although forefathers have done largely Work, and a collection of mildew-resistance related gene is obtained, but the mesh for carrying Pm21 gene can be seldom navigated in these genes Mark chromosome segment.Although mildew-resistance process (the Cao et of the genes such as stpk-V, DvPP2C participation Pm21 gene mediated al.Serine/threonine kinase gene Stpk-V,a key member of powdery mildew resistance gene Pm21,confers powdery mildew resistance in wheat.Proc Natl Acad Sci USA.2011,108:7727-7732；He et al.Comparative mapping of powdery mildew resistance gene Pm21and functional characterization of resistance- Related genes in wheat.Theor Appl Genet.2016,129:819-829), but gene silencing analytical table Bright, there are macroscopic white powder disease symptoms in the disease-resistant material that the silencing of these genes not will lead to the gene containing Pm21, this says It is bright that there is presently no be cloned into real Pm21.

Plant disease-resistant albumen plays double action in terms of identification pathogenic factor and starting defence response.From the plant being sequenced In object genome, scientific worker predicts a large amount of disease resistance gene analogs (RGA), and wherein nucleotide binding site-is rich in bright It is main Types that propylhomoserin, which repeats (NBS-LRR) class disease-resistant gene,.It is all mainly containing spiral shell in the wheat resistance genes cloned The NBS-LRR class disease-resistant gene of circumvolution song (CC) structural domain, i.e. CC-NBS-LRR class disease-resistant gene.But it is directed to Pm21 and PmV The research of gene, researcher are concentrated mainly on various kinases and transcription factor, and the research to CC-NBS-LRR class disease-resistant gene Seldom.The present invention uses wheat crops-two fringe false bromegrass comparative genomics, Direct Cloning haynaldia villosa RGA, integrated use heredity The technological means such as mapping, physical mapping, mutant research, virus induced gene silencing analysis and Transient Expression analysis, it was demonstrated that Candidate gene DvRGA-1 obtained can significantly improve wheat to the resistance of powdery mildew.In turn from the another of carrying PmV gene The DvRGA-2 gene with DvRGA-1 very high homology has been cloned in the haynaldia villosa in source (former Soviet Union), and confirms DvRGA-2 Wheat be can be improved to the resistance of powdery mildew.In conclusion the present invention discloses haynaldia villosa mildew-resistance gene DvRGA-1 for the first time With the clone of DvRGA-2, and on this basis propose national inventing patent application.

Summary of the invention

It is an object of the invention to disclose the mildew-resistance gene DvRGA-1 and DvRGA- of the haynaldia villosa of two separate sources 2 gene order.The genomic dna sequence of DvRGA-1 gene is SEQ ID NO.1, and corresponding cDNA sequence is SEQ ID NO.2, the amino acid sequence of coding are SEQ ID NO.3.The genomic dna sequence of DvRGA-2 gene is SEQ ID NO.4, right The cDNA sequence answered is SEQ ID NO.5, and the amino acid sequence of coding is SEQ ID NO.6.Functional analysis shows DvRGA-1 Gene and DvRGA-2 gene can significantly improve wheat to the resistance of powdery mildew.

Haynaldia villosa mildew-resistance gene DvRGA-1, genomic dna sequence are SEQ ID NO.1, and cDNA sequence is SEQ ID NO.2, the protein sequence of coding are SEQ ID NO.3.

Haynaldia villosa mildew-resistance gene DvRGA-2, genomic dna sequence are SEQ ID NO.4, and cDNA sequence is SEQ ID NO.5, the protein sequence of coding are SEQ ID NO.6.

The haynaldia villosa mildew-resistance gene DvRGA-1 can be applied to genetic improvement wheat breed, and to improve its powdery mildew anti- Property.

The haynaldia villosa mildew-resistance gene DvRGA-2 can be applied to genetic improvement wheat breed, and to improve its powdery mildew anti- Property.

DvRGA-1 and DvRGA-2 gene is derived from the gene of different haynaldia villosas, and the two sequence height is similar, but also deposits In different, the two belongs to homologous gene, this shows in DvRGA-1 homologous gene, and the change of partial dna sequence is not It will affect the effect of its mildew-resistance.In natural population, the haynaldia villosa of most separate sources all has good powdery mildew Therefore resistance easily speculates the homologous gene of the DvRGA-1 and DvRGA-2 gene from these disease-resistant haynaldia villosas and through part DvRGA-1 the and DvRGA-2 gene that DNA sequence dna replaces also has powder mildew resistance, they are in wheat anti-powdery mildew breeding With application value.

Beneficial effects of the present invention:

The mildew-resistance gene DvRGA-1 and DvRGA-2 for the haynaldia villosa that the present invention announces are to report for the first time, DvRGA-1 Expression with DvRGA-2 gene can significantly improve wheat to the resistance of powdery mildew, using DvRGA-1 and DvRGA-2 gene as mesh Gene carry out the transgenosis of wheat, wheat breed can be improved, improve wheat breed to the resistance of powdery mildew.

Detailed description of the invention

Fig. 1: DvRGA-1, the structure of DvRGA-2 gene.There are three exon (1-407,505- for DvRGA-1 gene tool 897,1770-3699) and two intrones (408-504,898-1769).DvRGA-2 gene tool there are three exon (1-407, 504-896,1769-3692) and two intrones (408-503,897-1768).Grey, black, grey parts respectively indicate volume CC structural domain, NBS structural domain, the LRR structural domain of code albumen.

Fig. 2: DvRGA-1, the chadogram of DvRGA-2 and other CC-NBS-LRR albuminoids.BRADI3G03874, BRADI3G03878, BRADI3G03882, BRADI3G03935 are two fringe false bromegrass ill-resistant protein, and PM2, PM3b, PM8 are wheat Mildew-resistance albumen, MLA10 are barley mildew-resistance albumen, and SR22, SR33, SR35, SR45, SR50 are the anti-stem rust egg of wheat White, LR1, LR10 are wheat leaf rust resistance albumen, and YR10 is wheat stripe rust resisting disease albumen, and RPM1, RPP13 are the disease-resistant egg of arabidopsis It is white.

Fig. 3: DvRGA-1, the genetic mapping of Pm21 gene and deletion mapping.Left figure is to utilize mildew-resistance haynaldia villosa and sense The F of powdery mildew haynaldia villosa DvSus-1 building₂The genetic map that mapping population is obtained, right figure are to be mutated using susceptible wheat 18 of raising The missing map of body Y18-S16 building.The corresponding molecular labeling of DvRGA-1 gene is 6VS-09.4b and 6VS-09.4.Vertical arrow Head indicates the centromere direction of chromosome.Dotted portion indicates the deleted segment in Y18-S16.Horizontal arrow indicates that chromosome is disconnected Point b1 and b2, and the chromosomal breakpoint FL0.45 and FL0.58 that find in the past, wherein FL0.58 and b1 substantially close to.Mark b Molecular labeling and correspondence markings be by according to the same Data mining come.Label with * is (SNP) Label.

Fig. 4: DvRGA-1, the quantitative fluorescent PCR (qPCR) of DvRGA-2 gene analyzes result.It is raised respectively with powdery mildew inoculation Wheat 18 and Yang Mai 22,0,6,12,24,48,72h, qPCR detection is raised in wheat 18 after sample time is followed successively by powdery mildew inoculation The transcription of DvRGA-1 gene and the transcription for raising DvRGA-2 gene in wheat 22.

Fig. 5: DvRGA-1, the silencing analysis of DvRGA-2 gene.A: powdery mildew spores are sprouted under microscope, mycelia is developed Figure, B: the lower blade phenotypic map of naked eyes.A: inoculation BSMV:00 (be free of foreign gene segment) to raise wheat 18 (carrying Pm21 gene) right According to b: wheat 18, c is raised in inoculation BSMV:DvRGA-1as (gene segment containing the DvRGA-1 being reversely inserted into): inoculation BSMV: DvRGA-1as's raises wheat 22 (carrying PmV gene).

Fig. 6: it raises wheat 18 and feels powdery mildew mutation type surface.Y18-S1,Y18-S2,Y18-S3,Y18-S4,Y18-S5,Y18- S6:6 plants independently obtain raise the susceptible mutant of wheat 18.It is susceptible to raise wheat 9 to raise wheat 18 as disease-resistant control when powdery mildew is identified Control.

Fig. 7: it raises wheat 18 and feels distribution and frequency of occurrence of the mutational site on DvRGA-1 protein in powdery mildew mutant. Ordinate is the number that specific mutation occurs, and abscissa is the amino acid sequence (from N-terminal to C-terminal) of DvRGA-1 albumen, abscissa Lower section be DvRGA-1 albumen structural domain: CC structural domain, NBS structural domain and LRR structural domain are that the small frame of grey is shown Conserved motifs (the Meyers et al.Receptor-like genes in the major resistance locus of report of lettuce are subject to divergent selection.Plant Cell.1998,11:1833–1846)。

Specific embodiment

1, the clone of Pm21 candidate gene DvRGA-1, sequence analysis and phylogenetic analysis

The present invention utilizes long range PCR technology, with P1 (5 '-GAAATAATGGGCACCCCTTCTTA CTA-3 ') and P2 (5 '-ATGCACTTACATCGTTAGTTACAACATC-3 ') are primer, the disease-resistant haynaldia villosa from Britain Camb botanical garden source (public, Chen et al.Development and molecular cytogenetic analysis of wheat- Haynaldia villosa 6VS/6AL translocation lines specifying resistance to Powdery mildew.Theor Appl Genet.1995,91:1125-1128) gene has been cloned in genome, due to The gene source in haynaldia villosa (Dasyprum vIllosum), and for disease resistance gene analog (Resistance GeneANalog), thus it is named as DvRGA-1.DNA sequencing discovery, the genomic DNA overall length of DvRGA-1 gene are 3699bp, Particular sequence is that there are three exon and two intrones (see Fig. 1), RT-PCR analyses for SEQ ID NO.1, DvRGA-1 gene tool Disclosing its cDNA sequence length with entire open reading frame is 2730bp, is SEQ ID NO.2, the protein of coding For 909aa, protein sequence is SEQ ID NO.3, encodes typical CC-NBS-LRR class ill-resistant protein.In the plant cloned In CC-NBS-LRR class ill-resistant protein, DvRGA-1 albumen and the sequence identity of wheat SR22, SR35, barley MLA10 are respectively 35.0%, 34.2%, 29.7%, phylogenetic analysis also discloses DvRGA2 albumen and these protein similarities are higher, and with gram The similitude of grand powdery mildew resistance gene in wheat coding albumen PM3, PM8, PM2 are lower (see Fig. 2).

2, the genetic mapping and physical mapping of DvRGA-1 gene and Pm21 gene

The present invention has found haynaldia villosa DvSus-1~DvSus-4 of sense powdery mildew from wild resource for the first time, and then with containing The disease-resistant haynaldia villosa in the Britain Camb botanical garden source of Pm21 gene hybridizes with susceptible DvSus-1, constructs F₂Genetic mapping group Body.The molecular labeling with polymorphism in anti-, sense haynaldia villosa parent is developed, with the molecular labeling 6VS-00.1 for being distributed in both ends And Xcfe164, from 10536 F₂64 exchange individuals are screened in single plant, and then carry out genetic mapping, by the Pm21 assignment of genes gene mapping Between molecular labeling 6VS-08.4 and 6VS-10b, i.e., area between the heredity of one 0.01cM.Genetic analysis shows candidate gene DvRGA-1 is in F₂It is isolated with Pm21 gene (see Fig. 3) in mapping population.

Physical mapping is carried out using the 18 mutant Y18-S16 of wheat that raises of the mutagenic obtained sense powdery mildew of EMS, finds susceptible mutation Deletion range also found, candidate gene simultaneously between molecular labeling 6VS-03 and 6VS-10.2 in body Y18-S16 DvRGA-1 also is located on the deletion where Pm21 gene (see Fig. 3).

Genetic mapping and physical mapping show that candidate gene DvRGA-1 is located at an extremely narrow Pm21 gene loci.

3, the transcription analysis of Pm21 candidate gene DvRGA-1

With powdery mildew induction raise wheat 18,0h, 6h, 12h, for 24 hours, 48h and 72h clip blade, extract total serum IgE, only After DNaseI digested genomic dna, quantitative fluorescent PCR (qPCR) analysis is carried out.The qPCR primer of DvRGA-1 gene is P3 (5 '- GGGAGCTGAGGCTTTACACT-3 ') and P4 (5 '-AGTACAACGGGTCGCAATGAT-3 '), it is wheat actin with reference to gene Gene, primer P5 (5 '-GCCGTGCTTTCCCTCTATG-3 ') and P6 (5 '-ATAAACTTCTGGTCCGAAAGC-3 ').qPCR The results show that DvRGA-1 gene is constitutive expression, after powdery mildew induces, expression is gradually increased, and is reached when to 48h Peak value (see Fig. 4).

4, the silencing analysis of Pm21 candidate gene DvRGA-1

With primer P7 (5 '-CTTGAATTCTGCGTCTAAGGGTGTTCGCT-3 ') and P8 (5 '- GGGGTCGACTTAAAGTAAAACTGGGACCACATTCATAGAAC-3 ') PCR amplification is carried out, obtain DvRGA2 gene end DNA fragmentation is connected into the BSMV γ carrier through same double digestion, after being sequenced, the γ of recombination after EcoRI and SalI double digestion Carrier, α carrier, β carrier (public) are linearized through Mlu I, Mlu I, Spe I respectively, after in-vitro transcription, three etc. Ratio mixing, formed recombinant virus BSMV:DvRGA-1as, frictional inoculation it is disease-resistant raise 18 second leaf of wheat.After virus infection 6 days, It is inoculated with powdery mildew, after 5 days, microexamination powdery mildew spores development after 10 days, visually observes the development of powdery mildew on blade Situation.The result shows that the silencing of DvRGA-1 gene can make the powdery mildew normal development raised on 18 blade of wheat go out a large amount of mycelia, with Occur macroscopic white powder disease symptoms (see Fig. 5) on blade afterwards, i.e., the silencing of DvRGA-1 gene can make disease-resistant to raise wheat 18 Morbidity completely, this shows that DvRGA-1 gene is the indispensable gene in the resistance of Pm21 gene mediated.

5, the Transient Expression analysis of Pm21 candidate gene DvRGA-1

First to expression vector pAHC25 (public, Christensen et al.Ubiquitin promoter- based vectors for high-level expression of selectable and/or screenable Marker genes in monocotyledonous plants.Transgenic Research.1996,5:213-218) into Multiple cloning sites MCS1 (Sma I-Not I-Mlu I-Spe I-Sac I), is substituted original gus gene by row transformation, is obtained PAHC25-MCS1 carrier.DvRGA-1 gene is connected into the pAHC25- through same double digestion after Sma I and Spe I double digestion MCS1 obtains recombinant plasmid pAHC25-DvRGA-1.Equivalent is carried out with the pAHC25 that can express GUS with pAHC25-DvRGA-1 to mix Close, using biolistic bombardment it is susceptible raise wheat No. 9, after biolistic bombardment 4h, be inoculated with powdery mildew, statistics haustorium refers to when infecting 48h Number.Mixed in equal amounts is carried out with empty carrier pAHC25-MCS1 and pAHC25 simultaneously, as negative control, is equally operated.As a result it shows Show, the haustorium index of control group is 64.5%, and the expression of DvRGA-1 gene can be such that haustorium index is reduced in test sample 21.2% (being shown in Table 1), this shows the resistance that the expression of DvRGA-1 gene is remarkably improved wheat to powdery mildew.

In conclusion genetic mapping, physical mapping, gene silencing analysis, Transient Expression analysis shows, candidate gene DvRGA-1 is played an important role during mildew-resistance.

The analysis of 1 Transient Expression of table

6, the mutation analysis of DvRGA-1 gene

About 10000 are handled using 0.8%EMS and raises 18 seed of wheat, obtain 6408 M₂Family.To M₂Family carries out white powder The dientification of bacteria, obtains 58 susceptible mutant, and susceptible mutant all shows as high sense powdery mildew with Adult plant in seedling stage (see Fig. 6). Wherein Y18-S16 is chromosome deficiency mutant.Mutant blade total serum IgE is extracted, after reverse transcription, with primer P9 (5 '- TTACCCGGGCTCACCCGTTGGACTTGGACT-3 ') and P10 (5 '- CCCACTAGTCTCTCTTCGTTACATAATGTAGTGCCT-3 ') from 57 mutant except Y18-S16 expand DvRGA- 1 gene, sequencing analysis show that in the 57 susceptible mutant surveyed, DvRGA-1 gene all has point mutation, wherein Y18- There are two the variations of base for DvRGA-1 gene tool in S35, Y18-S43, remaining is all single base variation.Raise the high frequency of wheat 18 Mutation shows that the resistance of Pm21 gene mediated controls for single copy gene, and the presence of the DvRGA-1 gene in all mutant Variation, this is an extreme small probability event, and therefore, DvRGA-1 gene is very likely exactly Pm21 gene.

In 57 susceptible mutant of all detections, DvRGA-1 gene co-exists in 59 variant sites, wherein 16 prominent Become the terminator codon caused in advance, there are 43 mutation to be related to amino acid variation.Due to the DvRGA- in Y18-S35, Y18-S43 1 gene tool not can determine that the variation in which site actually causes function to be lost, in subsequent analysis not there are two the variation of base Including statistics, so, it only analyzes 40 and is related to the mutation of amino acid variation.In this 40 mutation, 8 are located at CC structure Domain, 18 are located at NBS structural domain, and 9 are located at LRR structural domain, remaining bonding pad between each structural domain.It is being related to NBS In structural domain 18 mutation, 14 are located at known conserved motifs (motif) (Meyers et al.Receptor-like genes in the major resistance locus of lettuce are subject to divergent Selection.Plant Cell.1998,11:1833-1846) (see Fig. 7).

7, the clone of DvRGA-2 gene and functional analysis

It is the mildew-resistance gene of the haynaldia villosa in former Soviet Union source in view of PmV gene, and as Pm21 gene, also is located at 6VS chromosome, thus it is speculated that homologous gene DvRGA-2 of the Pm21 candidate gene DvRGA-1 in the haynaldia villosa in former Soviet Union source can also There can be mildew-resistance.Therefore, the present invention is with primer P9 (5 '-TTACCCGGGCTCACCCGTTGGACTTGGACT-3 ') Round pcr is directly utilized with P10 (5 '-CCCACTAGTCTCTCTTCGTTACATAATGTAGTGCCT-3 '), from carrying PmV base It can be expanded in former Soviet Union's haynaldia villosa of cause and obtain DvRGA-2 gene, genomic DNA overall length is 3692bp (SEQ ID NO.4), CDNA sequence is 2724bp (SEQ ID NO.5), and the protein of coding is 907aa (SEQ ID NO.6).DvRGA-2 gene Consensus dna sequence with DvRGA-1 gene is 96.4%, and the sequence identity of the two coding protein is 92.8%.With heavy The recombinant virus BSMV:DvRGA-1as infection carrying PmV gene of DvRGA-1 gene raises wheat 22 in Mo Yangmai 18, it has been found that, The silencing of DvRGA-2 gene can promote the development of powdery mildew, and form macroscopic powdery mildew symptom (see Fig. 6).Moment table Up to analysis shows that, DvRGA-2 gene raises the expression in wheat No. 9 haustorium can be made to be exponentially decreased to 19.8% (control group susceptible Haustorium index be 64.5%) (be shown in Table 1), it is seen that DvRGA-2 gene also can be improved wheat pair as DvRGA-1 gene The resistance of powdery mildew.

SEQUENCE LISTING

<110>Jiangsu University

<120>haynaldia villosa mildew-resistance gene DvRGA-1, DvRGA-2 and its application

<160>6

<170> Patent In version 3.3

<210>SEQ ID NO.1

<211>3699

<212>DNA

<213>haynaldia villosa (Dasyprum villosum)

<220>

<221>DvRGA-1 gene (genomic DNA)

<222>(1) ..(3699)

<400>1

atgtctgcac cggtcgtcag cgccaccatg ggggcgatga accccctcat cggcaagctc 60

gccgcactga ttggtgacga gtacaagaaa ctcacagggg tgaggagaca ggcctccttc 120

ctcaaggatg agcttagcgc catgaaagct ctccttgaga agcttgagct catggatgaa 180

ctggatccct tggccaagaa ctggagggat tatgtccggg agatgtccta cgacatggag 240

aattgcattg atgacttcat gcgagacctt ggaggtgccg atgcaaagac gggctttatc 300

aagaagacgg ctaaacgtct caagacgttg cggaagcgtc atcgtattgc tgatcggatg 360

gaagagctca aggggcttgc tttgcaagca aatgaacgac gcatgaggta caaatatgcc 420

ttcttttaat ataggagctc cttgctcgca attttatgca catgaaaccc ataaatatcc 480

agtttttctt gttccgatgc aaaggtacaa gattgatgat tgcgccaatt ctaccaatcg 540

tgtcgttccc atcgatactc ggatgttggc aatctacaag caggcaacgg ggcttgttgg 600

tattgatggc ccaaagaaag agcttgtaag ttggttgaca gatactcagg aaaaactcaa 660

ggtggtggct attgttggat ttggaggcct tggtaaaact acacttgcca aacaagtata 720

tgatacgatt ggagggcaat tcagctgtaa aatatttttc tctgtttctc aaagacctga 780

tatgtcaagc ctccttcgtg gtctccaatc ggagcttaat atggaagagg agttaactca 840

gcctcacgag gtgcaacaca tcattggccg tcttagagaa tatctcacac ataagaggta 900

cttattttgt tcgaattgac acttgtgcac ttgagtagtt atagtataga ggtttagagg 960

agtgtttctt gaatgggcaa catgcacaca tagttctgac actcccaatg ccttgaatcc 1020

cattccaccc atgctcgacg ccgtgttgtc atgggctcct gctctagctt gtcggaaccg 1080

aagatgggag agaaagagga tttttttttt tttgccaaga aaatttgatt gatgtggaat 1140

tagaagtcag tagaaagtaa aatggaaaat ccatagaata aacaaggcgg cactagcagg 1200

aagaatggca tgatcttcgt ttgaggtaag taaaactacc aggccgagaa aatatgcaaa 1260

ctttagatgg gggtatgaaa gggagaaaat tgatacttaa ttatttctcc attgagtatg 1320

taattgaaga tgaattagag agttacagaa aaaattatgg gccatctatc agatattact 1380

tgttatagat tgatcgattt ctaagattag ttggatccaa gaaaattggt acctgtatgg 1440

gtacataaat ctgcgggctc atgtcacttt cttttatggt ataatagaaa catgacgtct 1500

tttttttcca gacgtttcta agattagtta aatttgtttt acagtgtttt aacacgtcac 1560

acattaatta acttagtcta tgctagaatg ttatgtgaac atgctaaata atgtaaggag 1620

tatcatcttc ttgcttgaat tcttgtttag ttcattcttt gtttgtgttt attactaggt 1680

ttcagtgctt tcatcttctt ttttgcgggg acagtgcttt catcttcaac ttactatcat 1740

atcataatat gtacaaaaaa attgaacagg taccttattg ttgttgatga cttgtggtat 1800

caatcaacat ggaatatcat gagttgtatc tttccagaag tcgggaatgg aagtagagta 1860

atagtaacta cacgagtgga ggatgtggct atttgggcat gtcgtgatga ccatgagtgt 1920

gtttatagaa tggaacccct caaagaacaa gactcaagaa tgctgttctg taatagagta 1980

tttggttccg gatatgcctg tccactgccg ttaaaaaaag tttcagatga aattttgaag 2040

aagtgtggag ggttgccact tgcaattatc actatagcta gtctattagc aagtcgtcaa 2100

gcaagatcag acgagtggga gagcataaga aattgtttgg gcgctaagct tgccataaat 2160

tccaccttgg aagagatgag gagtatactg aaccttagct acatgcatct tcctcttcat 2220

ctccgtccat gtctcctgta ctttggcatg tatccagaag acaaaattat caggaggcgt 2280

gacatggttc tacagtgggt agccgaaggc tttgtcaata attctcatgg atctaatcta 2340

gaggatgttg cagagagtta tttcaatgag cttatcaata gaagtctaat tcagcctgga 2400

gaatccatag atggaaagat tgagtcttac aaagtacacg atatgatgct tgatttgatc 2460

ctcagcaagt gtgcagaaaa taattttata agtgtggcat ataattgtga agacgtggca 2520

agaatgcatg gccgcgaata caaggtccgt agattgtcct tgacttcaag tgctaacgat 2580

gcaacatcag aaaacattca tactagcatg caacaaattc gctcattttc atgctttgga 2640

gagcctaaat acacacctcc tcttttgcta tttaaatacc ttcgggtgct agtgtttata 2700

tcctcagacg cgtttggtcc gatagtggac cttactgcta ttggtcaatt gtttcagcta 2760

aggtatgtca aggtttctgc ttcatacgga atagattttc ctaccgaatt tcgcaagctt 2820

gttcatttgg agacgctgga agtatctggt ttttcaccaa gcatcccgtc agatattgtt 2880

tgcttgccac ggttatctcg tctgatcctt ccgtgtctta cacgtcttcc tcaagggatt 2940

gccaacataa aatcattgcg tgcattgcac tgtatggagc acatctcgct agaggatatt 3000

aatggccttg gcgagctgac cagtctgagg gagctgaggc tttacactaa aatggtggcg 3060

ggtgaagttg atgctttggt atccctaatt ggaaagctcc atgacctaaa atacctcgcg 3120

gtctctgttg agtcttctaa acatcattgc gacccgttgt actcattatc aaaccctcct 3180

ctccatatcg aggaacttga tctgtacggg tggacactga agagagttcc cacatggatt 3240

ggtgacctcc atttccttcg gatcctggat ttgtgtgtct acaacttgtt gaacgacgag 3300

gttcatgttg tgggaaatct tccctgcctc gtccatctgc gtctaagggt gttcgctgaa 3360

ggcggggccg taatctgcac gggcttattc caagtcctga aagtccttcg tctcttctct 3420

catgatgtgg aagacatgca gtttcagata gggctaatgc ccagcctgcg acagctcact 3480

ctagaagtaa ataatggctg gggtggtgct gtgcctcgag gcatggagca cctattggcc 3540

ctcgatcaca tctctgtatt tgccagacgc ggcgtcaatc accgtgatgt cgagtctgcc 3600

ttcagaagcg tcgtcgatgt gcacccaaga caaccttcct tagaaataat acctgatgtt 3660

cccctcagtt ctatgaatgt ggtcccagtt ttactttaa 3699

<110>Jiangsu University

<210>SEQ ID NO.2

<211>2730

<212>DNA

<213>haynaldia villosa (Dasyprum villosum)

<220>

<221>DvRGA-1 gene (cDNA)

<222>(1) ..(2730)

<400>2

atgtctgcac cggtcgtcag cgccaccatg ggggcgatga accccctcat cggcaagctc 60

gccgcactga ttggtgacga gtacaagaaa ctcacagggg tgaggagaca ggcctccttc 120

ctcaaggatg agcttagcgc catgaaagct ctccttgaga agcttgagct catggatgaa 180

ctggatccct tggccaagaa ctggagggat tatgtccggg agatgtccta cgacatggag 240

aattgcattg atgacttcat gcgagacctt ggaggtgccg atgcaaagac gggctttatc 300

aagaagacgg ctaaacgtct caagacgttg cggaagcgtc atcgtattgc tgatcggatg 360

gaagagctca aggggcttgc tttgcaagca aatgaacgac gcatgaggta caagattgat 420

gattgcgcca attctaccaa tcgtgtcgtt cccatcgata ctcggatgtt ggcaatctac 480

aagcaggcaa cggggcttgt tggtattgat ggcccaaaga aagagcttgt aagttggttg 540

acagatactc aggaaaaact caaggtggtg gctattgttg gatttggagg ccttggtaaa 600

actacacttg ccaaacaagt atatgatacg attggagggc aattcagctg taaaatattt 660

ttctctgttt ctcaaagacc tgatatgtca agcctccttc gtggtctcca atcggagctt 720

aatatggaag aggagttaac tcagcctcac gaggtgcaac acatcattgg ccgtcttaga 780

gaatatctca cacataagag gtaccttatt gttgttgatg acttgtggta tcaatcaaca 840

tggaatatca tgagttgtat ctttccagaa gtcgggaatg gaagtagagt aatagtaact 900

acacgagtgg aggatgtggc tatttgggca tgtcgtgatg accatgagtg tgtttataga 960

atggaacccc tcaaagaaca agactcaaga atgctgttct gtaatagagt atttggttcc 1020

ggatatgcct gtccactgcc gttaaaaaaa gtttcagatg aaattttgaa gaagtgtgga 1080

gggttgccac ttgcaattat cactatagct agtctattag caagtcgtca agcaagatca 1140

gacgagtggg agagcataag aaattgtttg ggcgctaagc ttgccataaa ttccaccttg 1200

gaagagatga ggagtatact gaaccttagc tacatgcatc ttcctcttca tctccgtcca 1260

tgtctcctgt actttggcat gtatccagaa gacaaaatta tcaggaggcg tgacatggtt 1320

ctacagtggg tagccgaagg ctttgtcaat aattctcatg gatctaatct agaggatgtt 1380

gcagagagtt atttcaatga gcttatcaat agaagtctaa ttcagcctgg agaatccata 1440

gatggaaaga ttgagtctta caaagtacac gatatgatgc ttgatttgat cctcagcaag 1500

tgtgcagaaa ataattttat aagtgtggca tataattgtg aagacgtggc aagaatgcat 1560

ggccgcgaat acaaggtccg tagattgtcc ttgacttcaa gtgctaacga tgcaacatca 1620

gaaaacattc atactagcat gcaacaaatt cgctcatttt catgctttgg agagcctaaa 1680

tacacacctc ctcttttgct atttaaatac cttcgggtgc tagtgtttat atcctcagac 1740

gcgtttggtc cgatagtgga ccttactgct attggtcaat tgtttcagct aaggtatgtc 1800

aaggtttctg cttcatacgg aatagatttt cctaccgaat ttcgcaagct tgttcatttg 1860

gagacgctgg aagtatctgg tttttcacca agcatcccgt cagatattgt ttgcttgcca 1920

cggttatctc gtctgatcct tccgtgtctt acacgtcttc ctcaagggat tgccaacata 1980

aaatcattgc gtgcattgca ctgtatggag cacatctcgc tagaggatat taatggcctt 2040

ggcgagctga ccagtctgag ggagctgagg ctttacacta aaatggtggc gggtgaagtt 2100

gatgctttgg tatccctaat tggaaagctc catgacctaa aatacctcgc ggtctctgtt 2160

gagtcttcta aacatcattg cgacccgttg tactcattat caaaccctcc tctccatatc 2220

gaggaacttg atctgtacgg gtggacactg aagagagttc ccacatggat tggtgacctc 2280

catttccttc ggatcctgga tttgtgtgtc tacaacttgt tgaacgacga ggttcatgtt 2340

gtgggaaatc ttccctgcct cgtccatctg cgtctaaggg tgttcgctga aggcggggcc 2400

gtaatctgca cgggcttatt ccaagtcctg aaagtccttc gtctcttctc tcatgatgtg 2460

gaagacatgc agtttcagat agggctaatg cccagcctgc gacagctcac tctagaagta 2520

aataatggct ggggtggtgc tgtgcctcga ggcatggagc acctattggc cctcgatcac 2580

atctctgtat ttgccagacg cggcgtcaat caccgtgatg tcgagtctgc cttcagaagc 2640

gtcgtcgatg tgcacccaag acaaccttcc ttagaaataa tacctgatgt tcccctcagt 2700

tctatgaatg tggtcccagt tttactttaa 2730

<110>Jiangsu University

<210>SEQ ID NO.3

<211>909

<212>PRT

<213>haynaldia villosa (Dasyprum villosum)

<220>

<221>DvRGA-1 albumen

<222>(1) ..(909)

<400>3

Met Ser Ala Pro Val Val Ser Ala Thr Met Gly Ala Met Asn Pro Leu

1 5 10 15

Ile Gly Lys Leu Ala Ala Leu Ile Gly Asp Glu Tyr Lys Lys Leu Thr

20 25 30

Gly Val Arg Arg Gln Ala Ser Phe Leu Lys Asp Glu Leu Ser Ala Met

35 40 45

Lys Ala Leu Leu Glu Lys Leu Glu Leu Met Asp Glu Leu Asp Pro Leu

50 55 60

Ala Lys Asn Trp Arg Asp Tyr Val Arg Glu Met Ser Tyr Asp Met Glu

65 70 75 80

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

85 90 95

Thr Gly Phe Ile Lys Lys Thr Ala Lys Arg Leu Lys Thr Leu Arg Lys

100 105 110

Arg His Arg Ile Ala Asp Arg Met Glu Glu Leu Lys Gly Leu Ala Leu

115 120 125

Gln Ala Asn Glu Arg Arg Met Arg Tyr Lys Ile Asp Asp Cys Ala Asn

130 135 140

Ser Thr Asn Arg Val Val Pro Ile Asp Thr Arg Met Leu Ala Ile Tyr

145 150 155 160

Lys Gln Ala Thr Gly Leu Val Gly Ile Asp Gly Pro Lys Lys Glu Leu

165 170 175

Val Ser Trp Leu Thr Asp Thr Gln Glu Lys Leu Lys Val Val Ala Ile

180 185 190

Val Gly Phe Gly Gly Leu Gly Lys Thr Thr Leu Ala Lys Gln Val Tyr

195 200 205

Asp Thr Ile Gly Gly Gln Phe Ser Cys Lys Ile Phe Phe Ser Val Ser

210 215 220

Gln Arg Pro Asp Met Ser Ser Leu Leu Arg Gly Leu Gln Ser Glu Leu

225 230 235 240

Asn Met Glu Glu Glu Leu Thr Gln Pro His Glu Val Gln His Ile Ile

245 250 255

Gly Arg Leu Arg Glu Tyr Leu Thr His Lys Arg Tyr Leu Ile Val Val

260 265 270

Asp Asp Leu Trp Tyr Gln Ser Thr Trp Asn Ile Met Ser Cys Ile Phe

275 280 285

Pro Glu Val Gly Asn Gly Ser Arg Val Ile Val Thr Thr Arg Val Glu

290 295 300

Asp Val Ala Ile Trp Ala Cys Arg Asp Asp His Glu Cys Val Tyr Arg

305 310 315 320

Met Glu Pro Leu Lys Glu Gln Asp Ser Arg Met Leu Phe Cys Asn Arg

325 330 335

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

340 345 350

Asp Glu Ile Leu Lys Lys Cys Gly Gly Leu Pro Leu Ala Ile Ile Thr

355 360 365

Ile Ala Ser Leu Leu Ala Ser Arg Gln Ala Arg Ser Asp Glu Trp Glu

370 375 380

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

385 390 395 400

Glu Glu Met Arg Ser Ile Leu Asn Leu Ser Tyr Met His Leu Pro Leu

405 410 415

His Leu Arg Pro Cys Leu Leu Tyr Phe Gly Met Tyr Pro Glu Asp Lys

420 425 430

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

435 440 445

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

450 455 460

Phe Asn Glu Leu Ile Asn Arg Ser Leu Ile Gln Pro Gly Glu Ser Ile

465 470 475 480

Asp Gly Lys Ile Glu Ser Tyr Lys Val His Asp Met Met Leu Asp Leu

485 490 495

Ile Leu Ser Lys Cys Ala Glu Asn Asn Phe Ile Ser Val Ala Tyr Asn

500 505 510

Cys Glu Asp Val Ala Arg Met His Gly Arg Glu Tyr Lys Val Arg Arg

515 520 525

Leu Ser Leu Thr Ser Ser Ala Asn Asp Ala Thr Ser Glu Asn Ile His

530 535 540

Thr Ser Met Gln Gln Ile Arg Ser Phe Ser Cys Phe Gly Glu Pro Lys

545 550 555 560

Tyr Thr Pro Pro Leu Leu Leu Phe Lys Tyr Leu Arg Val Leu Val Phe

565 570 575

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

580 585 590

Gln Leu Phe Gln Leu Arg Tyr Val Lys Val Ser Ala Ser Tyr Gly Ile

595 600 605

Asp Phe Pro Thr Glu Phe Arg Lys Leu Val His Leu Glu Thr Leu Glu

610 615 620

Val Ser Gly Phe Ser Pro Ser Ile Pro Ser Asp Ile Val Cys Leu Pro

625 630 635 640

Arg Leu Ser Arg Leu Ile Leu Pro Cys Leu Thr Arg Leu Pro Gln Gly

645 650 655

Ile Ala Asn Ile Lys Ser Leu Arg Ala Leu His Cys Met Glu His Ile

660 665 670

Ser Leu Glu Asp Ile Asn Gly Leu Gly Glu Leu Thr Ser Leu Arg Glu

675 680 685

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

690 695 700

Ser Leu Ile Gly Lys Leu His Asp Leu Lys Tyr Leu Ala Val Ser Val

705 710 715 720

Glu Ser Ser Lys His His Cys Asp Pro Leu Tyr Ser Leu Ser Asn Pro

725 730 735

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

740 745 750

Val Pro Thr Trp Ile Gly Asp Leu His Phe Leu Arg Ile Leu Asp Leu

755 760 765

Cys Val Tyr Asn Leu Leu Asn Asp Glu Val His Val Val Gly Asn Leu

770 775 780

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

785 790 795 800

Val Ile Cys Thr Gly Leu Phe Gln Val Leu Lys Val Leu Arg Leu Phe

805 810 815

Ser His Asp Val Glu Asp Met Gln Phe Gln Ile Gly Leu Met Pro Ser

820 825 830

Leu Arg Gln Leu Thr Leu Glu Val Asn Asn Gly Trp Gly Gly Ala Val

835 840 845

Pro Arg Gly Met Glu His Leu Leu Ala Leu Asp His Ile Ser Val Phe

850 855 860

Ala Arg Arg Gly Val Asn His Arg Asp Val Glu Ser Ala Phe Arg Ser

865 870 875 880

Val Val Asp Val His Pro Arg Gln Pro Ser Leu Glu Ile Ile Pro Asp

885 890 895

Val Pro Leu Ser Ser Met Asn Val Val Pro Val Leu Leu

900 905

<110>Jiangsu University

<210>SEQ ID NO.4

<211>3692

<212>DNA

<213>haynaldia villosa (Dasyprum villosum)

<220>

<221>DvRGA-2 gene (genomic DNA)

<222>(1) ..(3692)

<400>4

atgtctgcac cggtcgtcag cgccaccatg ggggcgatga accccctcat cggcaagctc 60

gccgcactga tgggtgacga gtacaagaaa ctcacagggg tgaggagaca ggcctccttc 120

ctcaaggatg agcttagcgc catgaaagct ctccttgaga agcttgagct catggatgaa 180

ctggatccct tggccaagaa ctggagggat catgtccggg agatgtccta cgacatggag 240

aattgcatcg atgacttcat gcgagacctt ggaggtgccg atgccaagat gggctttatc 300

aagaagacgg ctaaacgtct caagaggttg cggaagcgtc atcgtattgc tgatcggatg 360

gaagagctca aggtgcttgc tttggaagca aatgagcgac gcatgaggta caaatatcct 420

tcttttaata taggagctcc tagctcgcaa ttttatgcac atgaaaccca taaatatcca 480

gtttttcttg ttccgatgca aaggtacaag attgatgatt gcgccaattc taccaatcgt 540

gtcgttccca tcgatactcg gatgttggca atctacaagc aggcaacggg gcttgttggt 600

attgatggcc caaagaaaga gcttgtaagt tggttgacag atactcaaga aaaactcaag 660

gtggtggcta ttgttggatt tggaggcctc ggtaaaacta cacttgccaa acaagtatat 720

gatacgattg gagggcaatt cagctgtaaa atatttttat cagtttctca aagacctgat 780

atgtcaagcc tccttcgtgg tctccaatcg gagtttaaga tgagagagga gttaactcat 840

gctcacgagg tgcaacacat cattggccgt cttagagaat atctcacaca taagaggtac 900

ttattctgtt cgaactgaca cttgtgcact tgagtagtta tagaggttta gaggagttgt 960

ttcttgaatg ggcaacacaa cacaatgttc tgacactccc aatgccttga atcccattcc 1020

gtccatgctc gacgccctcc gtgttgttcg tgggctcctg ctctggcttg tcggaaccga 1080

agatgggaga gaaagaggct ttttttcaag aaaatttgat tgatgtggaa ttagaagtca 1140

gtagaaagta aaatagaaaa tccatagaat aaacaaggcg ccactagcag aaagaatggc 1200

atgatcttca tttgagttaa gtaaaactac caggccgaga agaaatgcaa actttagatg 1260

gggagtatga aagggagaaa attgacactt aatttgctat tatttctcca ttgagtatgt 1320

aattgaagat gaattagaga gttacagaaa aaattatggg ccatctatca gatattactt 1380

attatagatt gatcgatttc taagattagt tggatccaag aaaattggta cctgtatggg 1440

tacataaatc tgcgggctca tgtcactttc ttttatggta taatagaaac atgacgtctt 1500

ttttttccag acgtttctaa gattagttaa atttgtttta cagtgtttta acacgtcaca 1560

cattaattaa cttagtctat gctagaatgt tatgtgaaca tgctaaataa tgtaaggagt 1620

atcatcttct tgcttgaatt cttgtttagt tcattctttg tttgtgttta ttactaggtt 1680

tcagtgcttt catcttcttt tttgcgggga cagtgctttc atcttcaact tactatcata 1740

tcataatatg tacaaaaaaa ttgaacaggt accttattgt tgttgatgac ttgtggtatc 1800

aatcaacatg gaatatcatg agttgtatct ttccagaagt cgggaatgga agtagagtaa 1860

tagtaactac acgagtggag gatgtggcta tttgggcatg tcgcgatgac catgagtgtg 1920

tttatagaat ggaacccctc aaagaacaag actcaagaat gttgttctgt aatagagtat 1980

ttggttccgg accctgccca ccgcacttaa aaaaagtttc agatgaaatt ttgaagaagt 2040

gtggagggtt gccacttgca gttatcacta tagctagtct gttagcaagt cgtcaagcaa 2100

gatcagagga cgagtgggag agcataagaa attgtttggg cgccaagttt gccataaatc 2160

ccaccttgga agagatgagg agtatactga accttagcta catgcatctt cctcttcatc 2220

tccgtccatg tctcctgtac tttggcatgt atccagaaga caaaatgatc aagaggcgtg 2280

acatggttct acagtgggta gccgaaggct ttatcaataa ttctgatgga tctgatctag 2340

aggatgttgc agagagttat ttcaatgagc ttatcaatag aagtctaatt cagcctggag 2400

aatccataga tggaaagatt gagtcttaca aagtacacga tatgatgctt gatttgatcc 2460

tcagcaagtg tgcagaaaat aattttatta gtgtggcata taattgtgaa gacgtggcaa 2520

gaatgcatgg ctgcgaatac aaggtccgta gattgtcctt gacttcaagt gctgacgatg 2580

caacatcaga gaacatccat actagcatgc gacaaattcg ctcattctca tgctttggag 2640

agcctaaata cacacctcct cttttgctat ttaaatacct tcgggtgcta ctgtttatat 2700

ccccacacag ttttggtcgg atagtggact tcactgctat tggtcaattg tttcagctaa 2760

ggtatctcaa ggtttctgcc gcatgcagaa taatttttcc tagcgttcgc aagcttgttc 2820

atttggagac gctggaaata tctggtttca cacaaagcat accgtcagat attgtctgct 2880

tgccacggtt gtctcgtctg atccttccgt ttcatacatg tcttcctcaa gggattgcca 2940

acataaaatc attgcgtgca ctgcactgta tggagaacat ctcgctagag gatattaatg 3000

gccttggcga gctgaccagt ctgagggagt tgaggctttc taaagtggtg gcgggtgaaa 3060

ttgatgcttt ggtatcccta attggaaagc tccatgacct aaaatacctc gcggtctctg 3120

ttgagtcttc taaacatcat tgtgatccga tatactcatt atccaaccct cctctccata 3180

tcgaggaact tgatctgttc gggtggacac taaagagagt tcccacatgg attggtgacc 3240

tccatttcct tcggatcctg gttttgtggg tcgacaactt gtcgaacgac gaggttcatg 3300

ttgtgggaaa tctgccctgc ctcgtccatc tgcgtctaag ggtgttcgct gaaggcgggg 3360

ccgtaatctg caccggctta ttccaagtcc tgaaaggcct ttgtctcttc tctcatgatg 3420

tggaagacat gcagtttcag atagggctaa tgcccagcct gcgacagctc actctagaag 3480

taaataatgg ctggggaggt gctgtgcctc gaggcatgga gcacctattg gccctcgatc 3540

acatctctgt attttccaga cgcggcgtca atcaccgtga tgtcgagtct gccttcagaa 3600

gcgtcgtcga tgtgcaccca agacgacctt ccttagaaat aagacctgat gttcccctca 3660

gttctatgaa tgtggtcaca gttgtacttt aa 3692

<110>Jiangsu University

<210>SEQ ID NO.5

<211>2724

<212>DNA

<213>haynaldia villosa (Dasyprum villosum)

<220>

<221>DvRGA-2 gene (cDNA)

<222>(1) ..(2724)

<400>5

atgtctgcac cggtcgtcag cgccaccatg ggggcgatga accccctcat cggcaagctc 60

gccgcactga tgggtgacga gtacaagaaa ctcacagggg tgaggagaca ggcctccttc 120

ctcaaggatg agcttagcgc catgaaagct ctccttgaga agcttgagct catggatgaa 180

ctggatccct tggccaagaa ctggagggat catgtccggg agatgtccta cgacatggag 240

aattgcatcg atgacttcat gcgagacctt ggaggtgccg atgccaagat gggctttatc 300

aagaagacgg ctaaacgtct caagaggttg cggaagcgtc atcgtattgc tgatcggatg 360

gaagagctca aggtgcttgc tttggaagca aatgagcgac gcatgaggta caagattgat 420

gattgcgcca attctaccaa tcgtgtcgtt cccatcgata ctcggatgtt ggcaatctac 480

aagcaggcaa cggggcttgt tggtattgat ggcccaaaga aagagcttgt aagttggttg 540

acagatactc aagaaaaact caaggtggtg gctattgttg gatttggagg cctcggtaaa 600

actacacttg ccaaacaagt atatgatacg attggagggc aattcagctg taaaatattt 660

ttatcagttt ctcaaagacc tgatatgtca agcctccttc gtggtctcca atcggagttt 720

aagatgagag aggagttaac tcatgctcac gaggtgcaac acatcattgg ccgtcttaga 780

gaatatctca cacataagag gtaccttatt gttgttgatg acttgtggta tcaatcaaca 840

tggaatatca tgagttgtat ctttccagaa gtcgggaatg gaagtagagt aatagtaact 900

acacgagtgg aggatgtggc tatttgggca tgtcgcgatg accatgagtg tgtttataga 960

atggaacccc tcaaagaaca agactcaaga atgttgttct gtaatagagt atttggttcc 1020

ggaccctgcc caccgcactt aaaaaaagtt tcagatgaaa ttttgaagaa gtgtggaggg 1080

ttgccacttg cagttatcac tatagctagt ctgttagcaa gtcgtcaagc aagatcagag 1140

gacgagtggg agagcataag aaattgtttg ggcgccaagt ttgccataaa tcccaccttg 1200

gaagagatga ggagtatact gaaccttagc tacatgcatc ttcctcttca tctccgtcca 1260

tgtctcctgt actttggcat gtatccagaa gacaaaatga tcaagaggcg tgacatggtt 1320

ctacagtggg tagccgaagg ctttatcaat aattctgatg gatctgatct agaggatgtt 1380

gcagagagtt atttcaatga gcttatcaat agaagtctaa ttcagcctgg agaatccata 1440

gatggaaaga ttgagtctta caaagtacac gatatgatgc ttgatttgat cctcagcaag 1500

tgtgcagaaa ataattttat tagtgtggca tataattgtg aagacgtggc aagaatgcat 1560

ggctgcgaat acaaggtccg tagattgtcc ttgacttcaa gtgctgacga tgcaacatca 1620

gagaacatcc atactagcat gcgacaaatt cgctcattct catgctttgg agagcctaaa 1680

tacacacctc ctcttttgct atttaaatac cttcgggtgc tactgtttat atccccacac 1740

agttttggtc ggatagtgga cttcactgct attggtcaat tgtttcagct aaggtatctc 1800

aaggtttctg ccgcatgcag aataattttt cctagcgttc gcaagcttgt tcatttggag 1860

acgctggaaa tatctggttt cacacaaagc ataccgtcag atattgtctg cttgccacgg 1920

ttgtctcgtc tgatccttcc gtttcataca tgtcttcctc aagggattgc caacataaaa 1980

tcattgcgtg cactgcactg tatggagaac atctcgctag aggatattaa tggccttggc 2040

gagctgacca gtctgaggga gttgaggctt tctaaagtgg tggcgggtga aattgatgct 2100

ttggtatccc taattggaaa gctccatgac ctaaaatacc tcgcggtctc tgttgagtct 2160

tctaaacatc attgtgatcc gatatactca ttatccaacc ctcctctcca tatcgaggaa 2220

cttgatctgt tcgggtggac actaaagaga gttcccacat ggattggtga cctccatttc 2280

cttcggatcc tggttttgtg ggtcgacaac ttgtcgaacg acgaggttca tgttgtggga 2340

aatctgccct gcctcgtcca tctgcgtcta agggtgttcg ctgaaggcgg ggccgtaatc 2400

tgcaccggct tattccaagt cctgaaaggc ctttgtctct tctctcatga tgtggaagac 2460

atgcagtttc agatagggct aatgcccagc ctgcgacagc tcactctaga agtaaataat 2520

ggctggggag gtgctgtgcc tcgaggcatg gagcacctat tggccctcga tcacatctct 2580

gtattttcca gacgcggcgt caatcaccgt gatgtcgagt ctgccttcag aagcgtcgtc 2640

gatgtgcacc caagacgacc ttccttagaa ataagacctg atgttcccct cagttctatg 2700

aatgtggtca cagttgtact ttaa 2724

<110>Jiangsu University

<210>SEQ ID NO.6

<211>907

<212>PRT

<213>haynaldia villosa (Dasyprum villosum)

<220>

<221>DvRGA-2 albumen

<222>(1) ..(907)

<400>6

Met Ser Ala Pro Val Val Ser Ala Thr Met Gly Ala Met Asn Pro Leu

1 5 10 15

Ile Gly Lys Leu Ala Ala Leu Met Gly Asp Glu Tyr Lys Lys Leu Thr

20 25 30

Gly Val Arg Arg Gln Ala Ser Phe Leu Lys Asp Glu Leu Ser Ala Met

35 40 45

Lys Ala Leu Leu Glu Lys Leu Glu Leu Met Asp Glu Leu Asp Pro Leu

50 55 60

Ala Lys Asn Trp Arg Asp His Val Arg Glu Met Ser Tyr Asp Met Glu

65 70 75 80

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

85 90 95

Met Gly Phe Ile Lys Lys Thr Ala Lys Arg Leu Lys Arg Leu Arg Lys

100 105 110

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

115 120 125

Glu Ala Asn Glu Arg Arg Met Arg Tyr Lys Ile Asp Asp Cys Ala Asn

130 135 140

Ser Thr Asn Arg Val Val Pro Ile Asp Thr Arg Met Leu Ala Ile Tyr

145 150 155 160

Lys Gln Ala Thr Gly Leu Val Gly Ile Asp Gly Pro Lys Lys Glu Leu

165 170 175

Val Ser Trp Leu Thr Asp Thr Gln Glu Lys Leu Lys Val Val Ala Ile

180 185 190

Val Gly Phe Gly Gly Leu Gly Lys Thr Thr Leu Ala Lys Gln Val Tyr

195 200 205

Asp Thr Ile Gly Gly Gln Phe Ser Cys Lys Ile Phe Leu Ser Val Ser

210 215 220

Gln Arg Pro Asp Met Ser Ser Leu Leu Arg Gly Leu Gln Ser Glu Phe

225 230 235 240

Lys Met Arg Glu Glu Leu Thr His Ala His Glu Val Gln His Ile Ile

245 250 255

Gly Arg Leu Arg Glu Tyr Leu Thr His Lys Arg Tyr Leu Ile Val Val

260 265 270

Asp Asp Leu Trp Tyr Gln Ser Thr Trp Asn Ile Met Ser Cys Ile Phe

275 280 285

Pro Glu Val Gly Asn Gly Ser Arg Val Ile Val Thr Thr Arg Val Glu

290 295 300

Asp Val Ala Ile Trp Ala Cys Arg Asp Asp His Glu Cys Val Tyr Arg

305 310 315 320

Met Glu Pro Leu Lys Glu Gln Asp Ser Arg Met Leu Phe Cys Asn Arg

325 330 335

Val Phe Gly Ser Gly Pro Cys Pro Pro His Leu Lys Lys Val Ser Asp

340 345 350

Glu Ile Leu Lys Lys Cys Gly Gly Leu Pro Leu Ala Val Ile Thr Ile

355 360 365

Ala Ser Leu Leu Ala Ser Arg Gln Ala Arg Ser Glu Asp Glu Trp Glu

370 375 380

Ser Ile Arg Asn Cys Leu Gly Ala Lys Phe Ala Ile Asn Pro Thr Leu

385 390 395 400

Glu Glu Met Arg Ser Ile Leu Asn Leu Ser Tyr Met His Leu Pro Leu

405 410 415

His Leu Arg Pro Cys Leu Leu Tyr Phe Gly Met Tyr Pro Glu Asp Lys

420 425 430

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

435 440 445

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

450 455 460

Phe Asn Glu Leu Ile Asn Arg Ser Leu Ile Gln Pro Gly Glu Ser Ile

465 470 475 480

Asp Gly Lys Ile Glu Ser Tyr Lys Val His Asp Met Met Leu Asp Leu

485 490 495

Ile Leu Ser Lys Cys Ala Glu Asn Asn Phe Ile Ser Val Ala Tyr Asn

500 505 510

Cys Glu Asp Val Ala Arg Met His Gly Cys Glu Tyr Lys Val Arg Arg

515 520 525

Leu Ser Leu Thr Ser Ser Ala Asp Asp Ala Thr Ser Glu Asn Ile His

530 535 540

Thr Ser Met Arg Gln Ile Arg Ser Phe Ser Cys Phe Gly Glu Pro Lys

545 550 555 560

Tyr Thr Pro Pro Leu Leu Leu Phe Lys Tyr Leu Arg Val Leu Leu Phe

565 570 575

Ile Ser Pro His Ser Phe Gly Arg Ile Val Asp Phe Thr Ala Ile Gly

580 585 590

Gln Leu Phe Gln Leu Arg Tyr Leu Lys Val Ser Ala Ala Cys Arg Ile

595 600 605

Ile Phe Pro Ser Val Arg Lys Leu Val His Leu Glu Thr Leu Glu Ile

610 615 620

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

625 630 635 640

Leu Ser Arg Leu Ile Leu Pro Phe His Thr Cys Leu Pro Gln Gly Ile

645 650 655

Ala Asn Ile Lys Ser Leu Arg Ala Leu His Cys Met Glu Asn Ile Ser

660 665 670

Leu Glu Asp Ile Asn Gly Leu Gly Glu Leu Thr Ser Leu Arg Glu Leu

675 680 685

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

690 695 700

Ile Gly Lys Leu His Asp Leu Lys Tyr Leu Ala Val Ser Val Glu Ser

705 710 715 720

Ser Lys His His Cys Asp Pro Ile Tyr Ser Leu Ser Asn Pro Pro Leu

725 730 735

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

740 745 750

Thr Trp Ile Gly Asp Leu His Phe Leu Arg Ile Leu Val Leu Trp Val

755 760 765

Asp Asn Leu Ser Asn Asp Glu Val His Val Val Gly Asn Leu Pro Cys

770 775 780

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

785 790 795 800

Cys Thr Gly Leu Phe Gln Val Leu Lys Gly Leu Cys Leu Phe Ser His

805 810 815

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

820 825 830

Gln Leu Thr Leu Glu Val Asn Asn Gly Trp Gly Gly Ala Val Pro Arg

835 840 845

Gly Met Glu His Leu Leu Ala Leu Asp His Ile Ser Val Phe Ser Arg

850 855 860

Arg Gly Val Asn His Arg Asp Val Glu Ser Ala Phe Arg Ser Val Val

865 870 875 880

Asp Val His Pro Arg Arg Pro Ser Leu Glu Ile Arg Pro Asp Val Pro

885 890 895

Leu Ser Ser Met Asn Val Val Thr Val Val Leu

900 905

Claims (4)

1. haynaldia villosa mildew-resistance geneDvRGA-1, it is characterised in that: genomic dna sequence is SEQ ID NO.1, cDNA sequence It is classified as SEQ ID NO.2, the protein sequence of coding is SEQ ID NO.3.

2. haynaldia villosa mildew-resistance geneDvRGA-2, it is characterised in that: genomic dna sequence is SEQ ID NO.4, cDNA sequence It is classified as SEQ ID NO.5, the protein sequence of coding is SEQ ID NO.6.

3. haynaldia villosa mildew-resistance gene described in claim 1DvRGA-1It is anti-that its powdery mildew is improved in genetic improvement wheat breed Application in property.

4. haynaldia villosa mildew-resistance gene described in claim 2DvRGA-2It is anti-that its powdery mildew is improved in genetic improvement wheat breed Application in property.