CN106676078A

CN106676078A - Selection of cucumber green mottle mosaic virus low virulent strain line and application of cucumber green mottle mosaic virus low virulent strain line in cross protection

Info

Publication number: CN106676078A
Application number: CN201710094854.4A
Authority: CN
Inventors: 李向东; 刘锦; 许帅; 田延平
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2017-02-22
Filing date: 2017-02-22
Publication date: 2017-05-17

Abstract

The invention relates to selection of a cucumber green mottle mosaic virus low virulent strain line and application of the cucumber green mottle mosaic virus low virulent strain line in cross protection. A wild type CGMMV-JN (GenBank No. KR232571) infectious clone is adopted as a basis, and mutation is introduced in fixed points in a genome of CGMMV through a site-directed mutation technique, so that four CGMMV mutants such as CP89A, CP114A, Rd68A, Rd869A and Rd1069A are obtained, wherein the pathogenicity of the mutants such as CP89A and Rd1069A is significantly reduced. The low virulent strain lines such as CP89A and Rd1069A can protect plants against being infected by CGMMV virulent strains.

Description

The screening and the application in cross protection of cucumber green mottle mosaic virus low virulent strain system

Technical field

The present invention relates to genetic engineering for plant virus resistance field, in particular it relates to cucumber green mottle mosaic virus The screening of (Cucumber green mottle mosaic virus, CGMMV) low virulent strain system and its answering in cross protection With.

Background technology

Cucumber green mottle mosaic virus (Cucumber green mottle mosaic virus, CGMMV) belong to tobacco Mosaic virus belongs to (Tobamovirus), mainly infects various ground family crops such as watermelon, cucumber, muskmelon, cucurbit, gives these works Safety in production on thing brings about great losses.Due to lacking the kind of anti-CGMMV immune or high in production, in the market is not again right The medicament of virosis special efficacy, thus the preventing and treating of CGMMV is extremely difficult.

Cross protection refers to after plant is infected by low virulent strain system, from the phenomenon that follow-up Virus strain infects, Achieved successfully through in the preventing and treating of many crop virosis.The limitation wide variety of key factor of cross protection is available weak Strain system is very little.At present, the research on CGMMV strains isolations is more, but on the research in CGMMV low virulent strains system very It is few.The present invention obtains two significantly reduced CGMMV low virulent strains systems of new pathogenicity by site-directed mutagenesis technique, attacks malicious real Checking is bright, and low virulent strain system CP89A and Rd1069A can effectively control infecting for CGMMV virulent strain departments.

The content of the invention

The invention provides a kind of screening and application of CGMMV low virulent strains system, based on the infectious clone of CGMMV, Introducing mutation is pinpointed in the gene of CGMMV by site-directed mutagenesis technique, the amino acid position of regulation and control CGMMV pathogenicities is specify that Point, obtains the weak virus mutants of CGMMV that pathogenicity is remarkably decreased.By determining cross protection of the low virulent strain system to virulent strain department Effect, obtains the low virulent strain system that can effectively protect plant to be infected from CGMMV virulent strain departments.

The present invention implement concrete technical scheme be：

A. the acquisition of mutant：Based on the infectious clone of the autonomous cucumber green mottle mosaic virus for building, for The 89th of its capsid protein (CP), 114 amino acids and depend on the 68th, 869 of RNA polymerase (RdRp) of RNA Mutant primer is designed with 1069 amino acids, using QuickChange^TMSite-directed mutagenesis kit is introduced in these sites and is mutated.

B. the screening of low virulent strain system：The mutant that will be obtained in a by agroinfiltration method is inoculated into host plant Ben's Cigarette, observes the change of symptom caused by these mutant, the mutant that screening pathogenicity is substantially reduced.

C. cross-protection is determined：10 days inoculation CGMMV virulent strain departments after inoculation low virulent strain system, observation low virulent strain system To the cross-protection of virulent strain department.

Using technical solutions according to the invention, following technique effect can be obtained：

1) the CGMMV low virulent strains system that two pathogenicities are substantially reduced is obtained.

2) low virulent strain system CP89A and Rd1069A can effectively protect plant infecting from CGMMV virulent strain departments.

Brief description of the drawings

Fig. 1 is the symptom of the 10th day after CGMMV virulent strain departments and mutant inoculation；

Fig. 2 is inoculated with the accumulating level of 10 Tian Shi CGMMV virulent strain departments and mutant RNA in tobacco；

Fig. 3 is inoculated with the accumulating level of 10 Tian Shi CGMMV virulent strain departments and mutant capsid protein (CP) in tobacco；

Fig. 4 intervals are 10 days, and low virulent strain ties up to 10 days protecting effects to tobacco plant after Challenge and method；

Fig. 5 is the accumulating level of CGMMV geneome RNAs in tobacco plant during cross protection is tested；

Fig. 6 is the accumulating level of CGMMV albumen in tobacco plant during cross protection is tested.

Specific embodiment

The specific embodiment of form, does further specifically to the above of the invention by the following examples It is bright, but this scope for being interpreted as above-mentioned theme of the invention should not be only limitted to following example.It is all based on the above of the present invention The technology realized belongs to the scope of the present invention.

Embodiment provided by the present invention, according to conventional laboratory conditions, the primer sequence such as following table employed in it：

F represents forward primer, and R represents reverse primer.The D and of CGMMV CP the 89th can be distinguished using primer in table The R of 114 sports A；RdRp E, the E of K and the 1069th of the 869th of the 68th is sported into A, the letter representative of overstriking The nucleotides of mutation.CP and ef1 α two are used for fluorescence quantitative PCR detection viral gene accumulating level to primer, and wherein EF1 α are plant Thing reference gene.

Embodiment 1：Rite-directed mutagenesis

With cucumber green mottle mosaic virus infectious clone pCaCGMMV as template, five sites are entered respectively using PCR Row mutation, polymerase used is Phusion exo+ polymerases (Finnzymes).

PCR reaction systems are as follows：

Reaction terminates, and 0.5 μ L Dpn I (20U/ μ L), 37 DEG C for the treatment of 2h are added in PCR primer, adds 125 μ L anhydrous The sodium acetate (pH=5.2) of ethanol and 5 μ L 3mol/L is mixed, -20 DEG C of precipitates overnights.13000r/min is centrifuged 10min, abandons Clearly, precipitation is placed in natural drying at room temperature after being washed with the ethanol of 1mL 75%, adds 10 μ L ddH₂O water back dissolvings, convert Escherichia coli Stbl3, mutant plasmid is through sequence verification.

Embodiment 2：Virus inoculation

By pCaCGMMV or mutant plasmid conversion Agrobacterium GV3101.Through bacterium colony PCR verify after, choose single spot be inoculated in containing In kanamycins (50 μ g/mL), rifamycin (50 μ g/mL), the LB liquid medium of tetracycline (50 μ g/mL).Take 500 μ L bacterium Liquid adds to 5mL 2- containing 10mmol/L (N- morpholines)-ethylsulfonic acid (MES) and 20 μm of ol/L acetosyringones (AS) and above-mentioned three In the LB culture mediums of kind antibiotic, 28 DEG C of shaken cultivations to exponential phase.Thalline is collected by centrifugation and 10mmol/ is resuspended in L MgCl₂, 10mmol/L MES, in 150 μm of ol/L AS, adjustment concentration makes its OD₆₀₀It is 0.5 or so, is stored at room temperature 3 hours. 5mL disposable syringes are taken, removes needle aspirate Agrobacterium bacterium solution, from Ben Shi cigarette (5-6 week old or 4-6 pieces true leaf) blade back of the body Face infiltrates.2 leaves of every plant of infiltration.The plant of infiltration is placed in culture (16 hours illumination/8 hour dark in 23 DEG C of illumination boxs Alternately).

Embodiment 3：Symptom Observation and virus concentration are detected

The symptom of inoculated plant is observed, viral gene and protein accumulation level are detected with quantitative fluorescent PCR and ELISA. The 10th day after inoculation, wild-type virus and mutant CP114A, Rd68A, Rd869A are in Ben Shi cigarette (Nicotiana Benthamiana obvious floral leaf and the symptom such as mottled) are caused on system leaf, and capsid protein the 89th and replicase RdRp the 1069 mutant hardly show symptom (Fig. 1) in Ben Shi cigarette.Be respectively designated as these weak virus mutants by inventor (wherein, the CP gene nucleotide series of CP89A are shown in Seq ID No.15, and its CP amino acid sequence is shown in Seq for CP89A and Rd1069A ID No.17；The RdRp gene nucleotide series of Rd1069A are shown in Seq ID No.16, and its RdRp amino acid sequence is shown in Seq ID No.18).Fluorescence quantitative PCR detection result shows, rna expression level of the two above mutant (strain) in tobacco leaf Significantly lower than wild-type virus and other mutant, viral accumulating level is about 10% (Fig. 2) of wild-type virus.ELISA is examined Survey result to show, viral accumulation of the two above strain in tobacco leaf is significantly lower than wild-type virus and other mutant (Fig. 3).

Embodiment 4：Cross-protection is determined

Choose the Ben Shi cigarette of 5 weeks or so, inoculation low virulent strain system CP89A and Rd1069A (protection inoculation).10 after protection inoculation Its inoculation virulent strain department.Each treatment 3 Ben Shi cigarette of inoculation, is repeated 3 times.

The 10th day investigation incidence after virulent strain department is inoculated with, finds only to be inoculated with the Ben Shi cigarette plant table of virulent strain department Now serious flower leaf paresthesia, the incidence of disease is 100%；And it is 8.3% and 0 to be inoculated with CP89A the and Rd1069A plant incidence of disease in advance (Fig. 4), illustrates that the protecting effect of two weak virus mutants is respectively 91.7% and 100% when interval being 10 days.

Fluorescence quantitative PCR detection result shows, during interval is the treatment of 10 days, disease in the tobacco of weak virus mutants protection The accumulating level of malicious RNA is significantly lower than the tobacco (Fig. 5) for not carrying out cross protection.Find that the Ben Shi cigarette of mutant protection is attacked simultaneously The concentration of malicious restrovirus particle is extremely low, so that Western blot cannot be detected (Fig. 6).

Result above shows that low virulent strain system CP89A and Rd1069A has preferable cross-protection to virulent strain department, Infecting for CGMMV virulent strain departments can effectively be protected the plants from.

<110>Shandong Agricultural University

<120>The screening and the application in cross protection of cucumber green mottle floral leaf low virulent strain system

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attacgcgta gccgtggagc tgagaagcga aa 32

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ctgtgaaggc tactgatgac gcgtctacag cc 32

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tcagtagcct tcacagcgtt aagcgactca gcag 34

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tccggcgttt tcgattagct ttaccgccac c 31

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cgaaaacgcc ggatacgcat cagttacaag cct 33

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ggagcgaccg ccgagattat agcgagggtc aat 33

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ggcggtcgct ccacaacccg gcactcc 27

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gtgcatgcaa ttcaaggaga aacctttgag gagacg 36

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cttgaattgc atgcacagta ttcacatcat tgtacccac 39

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cagactcaag cgggaaga 18

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aagccctatc gtaaacatca 20

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tggtgtcctc aagcctggta t 21

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<212>DNA

<213>Artificial sequence

<400>14

acgcttgaga tccttaaccg c 21

<210>15

<211>486

<212>DNA

<213>Cucumber green mottle viral capsid proteins（Coat Ptotein）Gene

<400>15

1 atggcttaca atccgatcac acctagcaaa cttattgcgt ttagtgcttc ttatgttccc

61 gtcaggactt tacttaattt tctagttgct tcacaaggta ccgctttcca gactcaagcg

121 ggaagagatt ctttccgcga gtccctgtct gcgttaccct cgtctgtcgt agatattaat

181 tctagattcc cagatgcggg tttttacgct ttcctcaacg gtcctgtgtt gaggcctatc

241 ttcgtttcgc ttctcagctc cacggatacg cgtaataggg tcattgaggt tgtagatcct

301 agcaatccta cgactgctga gtcgcttaac gctgtaaagc gtactgatga cgcgtctacg

361 gccgctaggg ctgagataga taatttaata gagtctattt ctaagggttt tgatgtttac

421 aatagggctt catttgaagc cgcgttttcg gtagtctggt cagaggctac cacctcgaaa

481 gcttag

<210>16

<211>4947

<212>DNA

<213>Cucumber green mottle varial polymerases（RNA-dependent RNA polymerase）Gene

<400>16

1 atggcaaaca ttaatgaaca aatcaacaac caacgtgacg ccgcggctag cgggagaaac

61 aatctcgtta gccaattggc gtcaaaaagg gtgtatgacg aggctgttcg ctcgttggat

121 catcaagaca gacgcccgaa aatgaacttt tctcgtgtgg tcagcacaga gcacaccagg

181 cttgtaactg atgcgtatcc ggagttttcg attagcttta ccgccaccaa gaactctgta

241 cactcccttg cgggtggtct gaggcttctt gaattggaat atatgatgat gcaagtgccc

301 tacggctcac cttgttatga catcggcggt aactatacgc agcacttgtt caaaggtaga

361 tcatatgtgc attgctgcaa tccgtgccta gatcttaaag atgttgcgag gaatgtgatg

421 tacaacgata tgatcacgca acatgtacag aggcacaagg gatctggcgg gtgcagacct

481 cttccaactt tccagataga tgcattcagg aggtacgata gttctccctg tgcggtcacc

541 tgttcagacg ttttccaaga gtgttcctat gattttggga gtggtaggga taatcatgcg

601 gtctcgttgc attcaatcta cgatatccct tattcttcga tcggacctgc tcttcatagg

661 aaaaatgtgc gagtttgtta tgcagccttt catttctcgg aggcactgct tttaggttcg

721 cctgtaggta atttaaatag tattggggct cagtttaggg tcgatggtga tgatgtgcat

781 tttcttttta gtgaagagtc tactttgcat tatactcata gtttagaaaa tatcaagtta

841 atcgtgatgc gtacttattt tcctgctgat gataggtttg tatatattaa ggagttcatg

901 gttaagcgtg tggatacttt tttctttagg ttggttaggg cagacacaca catgcttcat

961 aaatctgtgg ggcactattc gaaatcgaag tctgagtact tcgcgctgaa tacccctccg

1021 atcttccaag ataaagccac gttttctgtg tggtttcctg aagcgaagcg caaggtgttg

1081 atacccaagt ttgaactttc gagattcctt tctgggaatg tgaaaatctc taggatgctt

1141 gtcgatgctg atttcgtcca taccattatt aatcacatta gcacgtatga taacaaggcc

1201 ttagtgtgga aaaatgttca gtcctttgtg gaatccatac gttcaagagt aattgtaaac

1261 ggagtttccg tgaaatctga gtggaatgta ccggttgatc agctcactga tatctcgttc

1321 tcgatattcc ttctcgtgaa ggttaggaag gtacagatcg agttaatgtc tgataaagtt

1381 gtaatcgagg cgaggggttt gcttcggagg ttcgcagaca gtcttaaatc tgccgtagaa

1441 ggactaggtg attgcgtcta tgatgctcta gttcaaaccg gctggtttga cacctctagc

1501 gacgaactga aagtattgct acctgaaccg tttatgacct tttcggatta tcttgaaggg

1561 atgtacgagg cagatgcaaa gatcgagaga gagagtgtct ctgagttgct cgcttccggt

1621 gatgatttgt tcaagaaaat cgatgagata agaaacaatt acagtggagt cgaatttgat

1681 gtagagaaat tccaagaatt ttgcaaggaa ctgaatgtta atcctatgct aattggccac

1741 gttatcgaag ctattttttc gcagaaggct ggggtaacag taacaggttt gggcacgctc

1801 tctcctgaga tgggcgcttc tgttgcgtta tccagtacct ctgtagatac atgtgaagat

1861 atggatgtaa ctgaagatat ggaggatata gtgttgatgg cggacaaggg tcattcttac

1921 atgtcccctg aaatggcgag atgggctgat gttaaatatg gcaacaacaa aggagctcta

1981 gtcgagtaca aagtcggaac ctcgatgact ttacctgcca cctgggcaga gaaaggtaag

2041 gctgttttac cgttgtcggg gatctgtgtg aggaaacccc aattttcgaa gccgattgat

2101 gaggaagatg acttgaggtt atcaaacatg aatttcttta aggtgagcga tctaaagttg

2161 aagaagacta tcactccagt cgtttacact gggaccattc gagagaggca aatgaagaat

2221 tatattgatt acttatcggc ctctcttggt tccacgctgg gtaatctgga gaggatcgtg

2281 cggagtgatt ggaatggtac tgaggagagt atgcaaacgt tcgggttgta tgactgcgaa

2341 aagtgcaagt ggttattgtt gccagccgag aagaagcacg catgggccgt ggttctggca

2401 agtgatgata ccactcgcat aatcttcctt tcatatgacg aatctggttc tcctataatt

2461 gataagaaaa attggaagcg atttgctgtc tgttccgaga ccaaagtcta tagtgtaatt

2521 cgtagtttag aggttctaaa taaggaagca atagtcgacc ccggggttca cataacatta

2581 gttgacggag tgccgggttg tggaaagacc gccgagatta tagcgagggt caattggaaa

2641 actgatctag tattgactcc cgggagggag gcggctgcta tgattaggcg gagagcctgc

2701 gccctgcaca agtcacctgt ggcaaccagt gacaacgtca gaactttcga ctcttttgtg

2761 atgaataaga aaatcttcaa gtttgacgct gtctatgttg acgagggtct gatggtccat

2821 acgggattac ttaattttgc gttaaagatc tcaggttgta aaaaggcctt cgtctttggt

2881 gatgctaagc aaatcccgtt tataaacaga gtcatgaatt ttgattatcc taaagagtta

2941 agaactttaa tagtcgataa tgtagagcgt aggtatgtca cccataggtg tcctagagat

3001 gtcactagtt ttcttagtac tatctataaa gccgctgtcg ctactactag tccggttgta

3061 cattctgtga gggcaattaa agtgtcaggg gccggtattc tgaggcctga gttgacaaag

3121 atcagaggaa agataataac gtttactcaa tctgataagc agtctttgat caagagtggg

3181 cacaatgatg tgaatactgt gcatgaaatt cagggagaaa cctttgagga gacggcagtt

3241 gtgcgtgcca ccccgactcc aataggtttg attgcccgtg attcaccaca cgtactagtg

3301 gccttaacta ggcacactaa ggcaatggtg tattatactg ttgtattcga tgcagttaca

3361 agtataatag cggatgtgga aaaggtcgat cagtcgatct tgaccatgtt tgctaccact

3421 gtgcctacca aatagcaatt aatgcagaac tcgctgtatg tccatcgtaa tattttcctc

3481 cctgtaagta aaacggggtt ttatacagac atgcaggagt tctacgatag atgccttcct

3541 gggaattcct tcgtactgaa tgatttcgat gccgtaacca tgcggttgag ggacaacgaa

3601 tttaacttac aaccttgtag gctaaccttg agtaatttag atccggtacc cgctttaatt

3661 aagaatgaag cgcagaattt tctgatcccc gttttgcgta cggcctgtga aaagccgcgc

3721 attccgggtc ttcttgagaa tcttgtagct atgataaaga ggaatatgaa tactcctgat

3781 ttagctggga ccgtagatat aactaacatg tcgatttcta tagtagataa cttcttttct

3841 tcttttgtta gggacgaggt tttacttgat cacttagatt gtgttagggc tagttccatt

3901 caaagttttt ctgattggtt ttcgtgtcag ccaacctcag cggttggcca gttagctaat

3961 ttcaatttca tagatttgcc tgcctttgat acttatatgc atatgattaa gaggcaaccc

4021 aagagtcggt tagatacttc gattcagtct gaatatccgg ccttgcaaac tattgtttat

4081 caccctaaag tggtaaatgc ggtttttggt ccggttttca agtatttgac caccaagttt

4141 cttagtatgg tagatagttc taagtttttc ttttacacta ggaaaaaacc agaagatctg

4201 caggaatttt tctcagatct ctcttcccat tctgattatg agattcttga gcttgatgtt

4261 tctaaatatg acaagtcgca atccgatttc catttctctg ttgagatggc aatttgggaa

4321 aaattggggc tggacgatat tttggcttgg atgtggtcta tgggtcacaa aagaactata

4381 ctgcaagatt tccaagccgg gataaagacg ctcatttact atcaacggaa gtctggtgat

4441 gtaactactt ttataggtaa tacctttatt atcgcagcgt gtgtagctag tatgttgccg

4501 ttagataagt gttttaaagc tagtttttgt ggtgatgatt cgctgatcta ccttcctaag

4561 ggcttagagt atcctgatat acaggctact gccaaccttg tttggaattt tgaggcgaaa

4621 cttttccgaa agaagtatgg ttacttctgc gggaagtata taattcatca tgccaatggc

4681 tgtattgttt accctgaccc tttaaagtta attagtaaat taggtaataa gagtcttgta

4741 gggtatgagc atgttgagga gtttcgtata tctctcctcg acgttgctca tagtttgttt

4801 aatggtgctt atttccattt actcgacgat gcaatccacg aattatttcc taatgctggg

4861 ggttgcagtt ttgtaattaa ttgtttgtgt aagtatttga gtgataagcg ccttttccgt

4921 agtctttaca tagatgtctc taagtaa

<210>17

<211>161

<212>PRT

<213>Cucumber green mottle viral capsid proteins（Coat Ptotein）

<400>17

1 METAlaTyrAsnProIleThrProSerLysLeuIleAlaPheSerAlaSerTyrValPro

21 ValArgThrLeuLeuAsnPheLeuValAlaSerGlnGlyThrAlaPheGlnThrGlnAla

41 GlyArgAspSerPheArgGluSerLeuSerAlaLeuProSerSerValValAspIleAsn

61 SerArgPheProAspAlaGlyPheTyrAlaPheLeuAsnGlyProValLeuArgProIle

81 PheValSerLeuLeuSerSerThrAspThrArgAsnArgValIleGluValValAspPro

101 SerAsnProThrThrAlaGluSerLeuAsnAlaValLysArgThrAspAspAlaSerThr

121 AlaAlaArgAlaGluIleAspAsnLeuIleGluSerIleSerLysGlyPheAspValTyr

141 AsnArgAlaSerPheGluAlaAlaPheSerValValTrpSerGluAlaThrThrSerLys

161 Ala

<210>18

<211>1647

<212>PRT

<213>Cucumber green mottle varial polymerases（RNA-dependent RNA polymerase）

<400>18

1 METAlaAsnIleAsnGluGlnIleAsnAsnGlnArgAspAlaAlaAlaSerGlyArgAsn

21 AsnLeuValSerGlnLeuAlaSerLysArgValTyrAspGluAlaValArgSerLeuAsp

41 HisGlnAspArgArgProLysMETAsnPheSerArgValValSerThrGluHisThrArg

61 LeuValThrAspAlaTyrProGluPheSerIleSerPheThrAlaThrLysAsnSerVal

81 HisSerLeuAlaGlyGlyLeuArgLeuLeuGluLeuGluTyrMETMETMETGlnValPro

101 TyrGlySerProCysTyrAspIleGlyGlyAsnTyrThrGlnHisLeuPheLysGlyArg

121 SerTyrValHisCysCysAsnProCysLeuAspLeuLysAspValAlaArgAsnValMET

141 TyrAsnAspMETIleThrGlnHisValGlnArgHisLysGlySerGlyGlyCysArgPro

161 LeuProThrPheGlnIleAspAlaPheArgArgTyrAspSerSerProCysAlaValThr

181 CysSerAspValPheGlnGluCysSerTyrAspPheGlySerGlyArgAspAsnHisAla

201 ValSerLeuHisSerIleTyrAspIleProTyrSerSerIleGlyProAlaLeuHisArg

221 LysAsnValArgValCysTyrAlaAlaPheHisPheSerGluAlaLeuLeuLeuGlySer

241 ProValGlyAsnLeuAsnSerIleGlyAlaGlnPheArgValAspGlyAspAspValHis

261 PheLeuPheSerGluGluSerThrLeuHisTyrThrHisSerLeuGluAsnIleLysLeu

281 IleValMETArgThrTyrPheProAlaAspAspArgPheValTyrIleLysGluPheMET

301 ValLysArgValAspThrPhePhePheArgLeuValArgAlaAspThrHisMETLeuHis

321 LysSerValGlyHisTyrSerLysSerLysSerGluTyrPheAlaLeuAsnThrProPro

341 IlePheGlnAspLysAlaThrPheSerValTrpPheProGluAlaLysArgLysValLeu

361 IleProLysPheGluLeuSerArgPheLeuSerGlyAsnValLysIleSerArgMETLeu

381 ValAspAlaAspPheValHisThrIleIleAsnHisIleSerThrTyrAspAsnLysAla

401 LeuValTrpLysAsnValGlnSerPheValGluSerIleArgSerArgValIleValAsn

421 GlyValSerValLysSerGluTrpAsnValProValAspGlnLeuThrAspIleSerPhe

441 SerIlePheLeuLeuValLysValArgLysValGlnIleGluLeuMETSerAspLysVal

461 ValIleGluAlaArgGlyLeuLeuArgArgPheAlaAspSerLeuLysSerAlaValGlu

481 GlyLeuGlyAspCysValTyrAspAlaLeuValGlnThrGlyTrpPheAspThrSerSer

501 AspGluLeuLysValLeuLeuProGluProPheMETThrPheSerAspTyrLeuGluGly

521 METTyrGluAlaAspAlaLysIleGluArgGluSerValSerGluLeuLeuAlaSerGly

541 AspAspLeuPheLysLysIleAspGluIleArgAsnAsnTyrSerGlyValGluPheAsp

561 ValGluLysPheGlnGluPheCysLysGluLeuAsnValAsnProMETLeuIleGlyHis

581 ValIleGluAlaIlePheSerGlnLysAlaGlyValThrValThrGlyLeuGlyThrLeu

601 SerProGluMETGlyAlaSerValAlaLeuSerSerThrSerValAspThrCysGluAsp

621 METAspValThrGluAspMETGluAspIleValLeuMETAlaAspLysGlyHisSerTyr

641 METSerProGluMETAlaArgTrpAlaAspValLysTyrGlyAsnAsnLysGlyAlaLeu

661 ValGluTyrLysValGlyThrSerMETThrLeuProAlaThrTrpAlaGluLysGlyLys

681 AlaValLeuProLeuSerGlyIleCysValArgLysProGlnPheSerLysProIleAsp

701 GluGluAspAspLeuArgLeuSerAsnMETAsnPhePheLysValSerAspLeuLysLeu

721 LysLysThrIleThrProValValTyrThrGlyThrIleArgGluArgGlnMETLysAsn

741 TyrIleAspTyrLeuSerAlaSerLeuGlySerThrLeuGlyAsnLeuGluArgIleVal

761 ArgSerAspTrpAsnGlyThrGluGluSerMETGlnThrPheGlyLeuTyrAspCysGlu

781 LysCysLysTrpLeuLeuLeuProAlaGluLysLysHisAlaTrpAlaValValLeuAla

801 SerAspAspThrThrArgIleIlePheLeuSerTyrAspGluSerGlySerProIleIle

821 AspLysLysAsnTrpLysArgPheAlaValCysSerGluThrLysValTyrSerValIle

841 ArgSerLeuGluValLeuAsnLysGluAlaIleValAspProGlyValHisIleThrLeu

861 ValAspGlyValProGlyCysGlyLysThrAlaGluIleIleAlaArgValAsnTrpLys

881 ThrAspLeuValLeuThrProGlyArgGluAlaAlaAlaMETIleArgArgArgAlaCys

901 AlaLeuHisLysSerProValAlaThrSerAspAsnValArgThrPheAspSerPheVal

921 METAsnLysLysIlePheLysPheAspAlaValTyrValAspGluGlyLeuMETValHis

941 ThrGlyLeuLeuAsnPheAlaLeuLysIleSerGlyCysLysLysAlaPheValPheGly

961 AspAlaLysGlnIleProPheIleAsnArgValMETAsnPheAspTyrProLysGluLeu

981 ArgThrLeuIleValAspAsnValGluArgArgTyrValThrHisArgCysProArgAsp

1001 ValThrSerPheLeuSerThrIleTyrLysAlaAlaValAlaThrThrSerProValVal

1021 HisSerValArgAlaIleLysValSerGlyAlaGlyIleLeuArgProGluLeuThrLys

1041 IleArgGlyLysIleIleThrPheThrGlnSerAspLysGlnSerLeuIleLysSerGly

1061 HisAsnAspValAsnThrValHisGluIleGlnGlyGluThrPheGluGluThrAlaVal

1081 ValArgAlaThrProThrProIleGlyLeuIleAlaArgAspSerProHisValLeuVal

1101 AlaLeuThrArgHisThrLysAlaMETValTyrTyrThrValValPheAspAlaValThr

1121 SerIleIleAlaAspValGluLysValAspGlnSerIleLeuThrMETPheAlaThrThr

1141 ValProThrLys***GlnLeuMETGlnAsnSerLeuTyrValHisArgAsnIlePheLeu

1161 ProValSerLysThrGlyPheTyrThrAspMETGlnGluPheTyrAspArgCysLeuPro

1181 GlyAsnSerPheValLeuAsnAspPheAspAlaValThrMETArgLeuArgAspAsnGlu

1201 PheAsnLeuGlnProCysArgLeuThrLeuSerAsnLeuAspProValProAlaLeuIle

1221 LysAsnGluAlaGlnAsnPheLeuIleProValLeuArgThrAlaCysGluLysProArg

1241 IleProGlyLeuLeuGluAsnLeuValAlaMETIleLysArgAsnMETAsnThrProAsp

1261 LeuAlaGlyThrValAspIleThrAsnMETSerIleSerIleValAspAsnPhePheSer

1281 SerPheValArgAspGluValLeuLeuAspHisLeuAspCysValArgAlaSerSerIle

1301 GlnSerPheSerAspTrpPheSerCysGlnProThrSerAlaValGlyGlnLeuAlaAsn

1321 PheAsnPheIleAspLeuProAlaPheAspThrTyrMETHisMETIleLysArgGlnPro

1341 LysSerArgLeuAspThrSerIleGlnSerGluTyrProAlaLeuGlnThrIleValTyr

1361 HisProLysValValAsnAlaValPheGlyProValPheLysTyrLeuThrThrLysPhe

1381 LeuSerMETValAspSerSerLysPhePhePheTyrThrArgLysLysProGluAspLeu

1401 GlnGluPhePheSerAspLeuSerSerHisSerAspTyrGluIleLeuGluLeuAspVal

1421 SerLysTyrAspLysSerGlnSerAspPheHisPheSerValGluMETAlaIleTrpGlu

1441 LysLeuGlyLeuAspAspIleLeuAlaTrpMETTrpSerMETGlyHisLysArgThrIle

1461 LeuGlnAspPheGlnAlaGlyIleLysThrLeuIleTyrTyrGlnArgLysSerGlyAsp

1481 ValThrThrPheIleGlyAsnThrPheIleIleAlaAlaCysValAlaSerMETLeuPro

1501 LeuAspLysCysPheLysAlaSerPheCysGlyAspAspSerLeuIleTyrLeuProLys

1521 GlyLeuGluTyrProAspIleGlnAlaThrAlaAsnLeuValTrpAsnPheGluAlaLys

1541 LeuPheArgLysLysTyrGlyTyrPheCysGlyLysTyrIleIleHisHisAlaAsnGly

1561 CysIleValTyrProAspProLeuLysLeuIleSerLysLeuGlyAsnLysSerLeuVal

1581 GlyTyrGluHisValGluGluPheArgIleSerLeuLeuAspValAlaHisSerLeuPhe

1601 AsnGlyAlaTyrPheHisLeuLeuAspAspAlaIleHisGluLeuPheProAsnAlaGly

1621 GlyCysSerPheValIleAsnCysLeuCysLysTyrLeuSerAspLysArgLeuPheArg

1641 SerLeuTyrIleAspValSerLys

Claims

1. a kind of the low virulent strain system CP89A and Rd1069A of cucumber green mottle mosaic virus (CGMMV), it is characterised in that can pass through Following steps are obtained：

(1) mutation is introduced in CGMMV genomes fixed point using round pcr, obtains mutant, inoculation host's screening obtains capsid egg Low virulent strain system CP89A and Rd1069A that white 89th and the amino acids of polymerase the 1069th are undergone mutation.

(2) low virulent strain system CP89A and Rd1069A can protect plant infecting from CGMMV virulent strain departments.

2. the low virulent strain described in claim 1 ties up to the application that preventing and treating CGMMV infects aspect.