CN103947461B - A method for obtaining virus resistance in scion varieties, RNA interference vector pCAMBIA2300-CP and transgenic method - Google Patents

Abstract

A kind of method of scion variety acquisition virus resistance and rna interference vector pCAMBIA2300-CP and transgenic method of making of the present invention belongs to plant transgenic technology and application thereof.(1) antiviral transgenic rootstock is prepared; (2) scion grafting is made to grow to described transgenic rootstock; It is characterized in that: described antiviral transgenic rootstock obtains for proceeding to rna interference vector, the target sequence of described rna interference vector is the gene fragment in target viral gene group.The test that method of the present invention is used for tomato scion variety acquisition virus resistance obtains good effect, for in the specific embodiment of tomato, the rna interference vector of structure is pCAMBIA2300-1A, pCAMBIA2300-2A, pCAMBIA2300-2B, pCAMBIA2300-3A, pCAMBIA2300-CP or pCAMBIA2300-CMV5.Present invention also offers a kind of transgenic method of improvement.The antiviral plant that aforesaid method obtains effectively can evade the potential safety hazard of genetically modified food, and can substantially increase the efficiency obtaining antiviral transformed variety.

Description

A method for obtaining virus resistance in scion varieties, RNA interference vector pCAMBIA2300-CP and transgenic method

In the first and second office action notices of the parent case, the examiner pointed out that there were multiple inventions in the parent case, which were not unitary, so the applicant decided to file a divisional application for the unprotected inventions in the parent case. This application is a divisional application of application number 201110315208.9 (name of invention: a method for making scion varieties acquire virus resistance and RNA interference vector and transgenic method).

technical field

The invention relates to plant transgenic technology and application thereof, in particular to a method for obtaining virus resistance in tomato scion varieties, an RNA interference carrier and a transgenic method.

Background technique

Plant virus is an important cause of yield decline and quality deterioration of fruit trees, vegetables, flowers and other horticultural plants. In agricultural production, virus disease is the second largest type of disease after fungi. Due to the difficulty of prevention and treatment, it is known as "plant cancer". known as. At present, the commonly used prevention and control measures for plant viruses include elimination of virus sources and traditional media, application of detoxification technology, chemical control, and breeding of disease-resistant varieties, among which breeding of disease-resistant varieties is the most effective and economical method to prevent and control virus diseases. Due to its high efficiency and specificity, the plant disease resistance technology based on RNA interference technology has shown considerable prospects in plant virus resistance breeding.

Gene silencing or RNA interference (RNAi) refers to the formation of double-stranded RNA (double-stranded RNA, dsRNA) structure, thereby specifically degrading homologous target gene mRNA and silencing it (FireA., XuS., MontgomeryM.K., et al. -811). Since its interference occurs at the post-transcriptional level of the target gene, it is also called post-transcriptional gene silencing (PTGS). Plant RNAi has the characteristics of specificity, high efficiency, systemicity and heritability. RNAi technology can be used for gene function research; or the special trait variants created by RNAi can be directly used for plant improvement. Among them, RNAi technology has achieved good research results in plant virus resistance. RNAi is a defense mechanism for exogenous nucleic acid (such as virus) invasion that is ubiquitous in eukaryotes. At the same time, existing studies have further shown that the cross-protection of viruses is due to the induction of RNA silencing produced by viruses (weak strains) and the degradation of pathogenicity. Sexual homologous virus (strong strain) results (FrankG.R., StuartA.M.andDavidC.B. GeneSilencingwithoutDNA:RNA-MediatedCross-Protectionbetween Viruses [J]. PlantCell, 1999, 11: 1207-1216). A common strategy for using RNAi to resist viruses is to select a sequence of a plant virus gene and construct it into a double-stranded RNA (hairpinRNA, hpRNA) or an intron-containing ihpRNA (intron) in the form of an inverted repeat sequence. -hairpinRNA) expression vector, which is integrated into the plant gene through gene gun, viral vector transfection or Agrobacterium-mediated transformation, expresses dsRNA in vivo, directly or indirectly induces the production of RNAi, and achieves the purpose of resisting the virus. In the study of the hairpin structure, it is believed that the silencing efficiency of the ihpRNA expression vector is higher than that of the hpRNA expression vector (HelliwellC, WaterhouseP. Constructs and methods for high-throughput gene silencing inplants [J]. Methods, 2003, 30 (4): 289-295).

There are many types and varieties of horticultural plants, and the offspring of horticultural plants reproduced through vegetative propagation are generally under the threat of viruses. At present, there are dozens to hundreds of varieties of vegetables, fruits and flowers cultivated in production. Almost every variety has several to dozens of viral diseases. Viral diseases inhibit the growth and fruiting of plants and reduce the yield of fruits. , quality, and even cause dead trees. Once plants are infected by viruses, they will be poisoned for life and suffer lasting damage. It is feasible to use genetic engineering technology to improve each variety and improve its anti-virus ability, but it will cost a lot of manpower and financial resources. Grafting is one of the methods of propagation of many horticultural plants. Most fruit trees and some vegetables are propagated by grafting. Grafting can not only maintain the excellent characters of the scion species, but also utilize the favorable characteristics of the rootstock. In the application of grafting propagation, generally different varieties of the same species use the same rootstock, and even some different species of the same genus also use the same rootstock. The multi-purpose situation of such a stock makes improving rootstock varieties more efficient than improving scion varieties alone.

The so-called systemic gene silencing in plants means that gene silencing signals such as dsRNA, aberrantRNA or siRNA can be transmitted over short distances through plasmodesmata, or over long distances through the vascular system in plants (BrosnanC.A., MitterN ., ChristieM., et al.Nucleargene silencing directs reception of long-distance mRNA silencing in Arabidopsis [J]. ProcNatlAcadSciUSA, 2007, 104 (37): 14741-14746), (KalantidisK., SchumacherH.T., AlexiadisT., etal. ,100(1):13-26), and induces systemic silencing in whole plants. DanielH. et al. (DanielH.C, FabioT.S., MiyaD.H, etal. Pattern formation via small RNAmobility[J].Genes&Dev, 2009,23:549-554) showed that small RNA can be used as a mobile signal molecule to participate in plant development regulation. There are different reports on the transmission direction of silencing signals in plants. Palauqui et al. (PalauquiJ.C., ElmayanT., PollienJ.M., etal.Systemicacquiredsilencing:transgene-specificpost-transcriptionalsilencingistransmittedbygraftingfromsilencedstockstonon-silencedscions[J].EMBOJ,1997,16(15):4738-4745.) Tobacco as a test material , found that the conduction of PTGS is unidirectional, that is, it can only be transmitted from the silent rootstock to the non-silenced scion; while Sonoda et al. (SonodaS. (1):1-8) Studies have shown that the conduction of PTGS is bidirectional, from rootstock to scion or from scion to rootstock, but the conduction efficiency of PTGS from scion to rootstock is significantly lower than that from rootstock to scion . The results of Li Ming et al. (Li Ming, Jiang Shiling, Wang Youqun, et al. Post-transcriptional silencing signals can be rapidly bidirectionally transmitted in Arabidopsis grafts[J]. Science Bulletin, 2006,51(2):142-147) showed that no matter When RNAi plants were used as rootstock or scion, the gene silencing signal could lead to the decrease of target gene mRNA in the corresponding wild-type scion or rootstock, indicating that the post-transcriptional silencing signal could be bidirectionally transmitted in Arabidopsis through the grafting surface. However, miRNA (microRNA) or amiRNA (artificialmiRNA) has not been found to be transmitted between grafts. Accordingly, the inventor speculates that the direction of systematic gene silencing may be related to species or detection levels or methods, but it is certain that transgenic methods can be used to obtain super The expressed siRNA-associated silencing signal can be transmitted up from the rootstock to the scion.

In plants, the first clues to systemic gene silencing came from tobacco grafting experiments. In 1997, Palauqui et al. (PalauquiJ.C., ElmayanT., PollienJ.M., et al.Systemicacquiredsilencing:transgene-specificpost-transcriptionalsilencingistransmittedbygraftingfromsilencedstockstonon-silencedscions[J].EMBOJ,1997,16(15):4738-4745) grafted a bud When transferred to another plant, the nitrate reductase gene of the plant was first silenced, and the gene was also silenced on the grafted shoots. The genes that were silenced in the shoots were the same as those in the rootstock, with sequence specificity. Sonoda et al. (SonodaS.andNishiguchiM.Grafttransmissionofpost-transcriptionalgenesilencing:targetspecificityforRNAdegradationistransmissiblebetweensilencedandnon-silencedplants, butnotbetweensilencedplants[J].PlantJ,2000,21(1):1-8) also reached a similar conclusion, and proved that after grafting , the PTGS acquired by the scion was maintained even in the absence of a silent rootstock.

In recent years, around the breeding of genetically modified organisms, my country has achieved international standards in the study of safety management and safety evaluation of genetically modified organisms, and has made remarkable progress. However, some people have always had doubts about the impact of the resistance selectable marker gene involved in genetic engineering on food safety, and the research of this project cleverly avoided this issue, because the systemic resistance acquired by the scion , derived from the silencing effect of small molecule RNA in the rootstock, so theoretically, the grafted transgene silent plant scion varieties will not contain resistance markers. In order to improve the ability of the scion variety to resist the target virus, if the purpose of enhancing the resistance of the scion variety to the target virus is achieved only by improving the rootstock after grafting, this will open up a new way for the variety improvement of horticultural grafted plants. But so far, in the research on the gene silencing effect of scion varieties obtained by grafting, the target genes are some genes of the plant itself, and there is no research report on the target of pathogenic bacteria or virus genes.

Contents of the invention

According to the gaps and demands in the above-mentioned fields, the present invention provides a method for enabling tomato plants to obtain the ability to resist viruses, and the method involves RNA interference vectors, improved transgenic methods, etc., and the anti-virus plants obtained by the method can effectively avoid genetically modified foods potential safety hazards, and can greatly improve the efficiency of obtaining anti-virus transgenic varieties.

A method for making scion varieties obtain virus resistance, the steps are as follows:

(1) prepare antiviral transgenic stock;

(2) grafting the scion to the transgenic rootstock to grow and develop;

It is characterized in that: the anti-virus transgenic rootstock is obtained by transferring RNA interference vector, and the target sequence of the RNA interference vector is a gene segment in the target virus genome.

The target virus refers to cucumber mosaic virus CMV, the scion variety and the transgenic rootstock are both tomato varieties, and the target sequence of the RNA interference vector is shown in SeqID No.1, 2, 3, 4, 5 or 6.

The RNA interference vector is pCAMBIA2300-1A, pCAMBIA2300-2A, pCAMBIA2300-2B, pCAMBIA2300-3A, pCAMBIA2300-CP or pCAMBIA2300-CMV5.

Described preparation antiviral transgenic stock, transgenic comprises the steps:

(1). Tomato sowing, (2). Cut cotyledon pre-cultivation, (3). Bacteriostatic screening culture of cotyledon after Agrobacterium tumefaciens engineering bacteria infection, (4). Promoting differentiation culture, (5). Rooting culture , characterized by:

The seeds of the tomato were soaked in sterile water for 5 to 8 hours before sowing; 20% sodium hypochlorite was sterilized for 10 minutes, washed with sterile water 5 times, each time for 5 minutes; germination was accelerated for two days in a 30°C incubator;

The pre-cultivation of the cut cotyledons is to cut the cotyledons before the true leaves appear for pre-cultivation;

The antibacterial screening culture: cotyledons infected by Agrobacterium tumefaciens and cultured in dark for 2 days, medium C: pH6.0, MediumBase+2mg/L trans-zeatin nucleoside+200mg/L Timentin+ 100mg/L kanamycin, light culture with leaf back facing up, transfer once every 10 days until green callus or bud point appears; transfer the explant with bud point into medium C1: pH6.0, MediumBase+ 1mg/L trans-zeatin nucleoside + 80mg/L kanamycin + 200mg/L timentin, cultivated for 10-14 days, until differentiation and budding;

The differentiation-promoting culture: the resistant buds screened in the antibacterial culture were transferred to the medium C3: pH6.0, MediumBase+0.5mg/L trans-zeatin nucleoside+1.5mg/L3-indoleacetic acid+ 80mg/L Kanamycin + 200mg/L Timentin;

The culture medium for rooting culture is: pH6.0, MediumBase+2mg/LIBA+200mg/L Timentin.

An RNA interference vector targeting the cucumber mosaic virus gene, characterized in that the target sequence of the RNA interference vector is shown in SeqID No. 1, 2, 3, 4, 5 or 6.

The RNA interference vector is pCAMBIA2300-1A, pCAMBIA2300-2A, pCAMBIA2300-2B, pCAMBIA2300-3A, pCAMBIA2300-CP or pCAMBIA2300-CMV5.

A bacterial strain or plant cell transformed with the above-mentioned RNA interference vector.

The strain refers to Agrobacterium tumefaciens strain EHA105.

The plant cells refer to explant precursor cells of transgenic tomato rootstocks.

A method for preparing transgenic tomato rootstock, comprising the steps of:

(1). Tomato seeding, (2). Cut cotyledon pre-cultivation, (3). Bacteriostatic screening culture of cotyledon after Agrobacterium tumefaciens engineering bacteria infection,

(4). Promoting differentiation culture, (5). Rooting culture, it is characterized in that:

The seeds of the tomato were soaked in sterile water for 5 to 8 hours before sowing; 20% sodium hypochlorite was sterilized for 10 minutes, washed with sterile water 5 times, each time for 5 minutes; germination was accelerated for two days in a 30°C incubator;

The pre-cultivation of the cut cotyledons is to cut the cotyledons before the true leaves appear for pre-cultivation;

The antibacterial screening culture: cotyledons infected by Agrobacterium tumefaciens and cultured in dark for 2 days, medium C: pH6.0, MediumBase+2mg/L trans-zeatin nucleoside+200mg/L Timentin+ 100mg/L kanamycin, light culture with leaf back facing up, transfer once every 10 days until green callus or bud point appears; transfer the explant with bud point into medium C1: pH6.0, MediumBase+ 1mg/L trans-zeatin nucleoside + 80mg/L kanamycin + 200mg/L timentin, cultivated for 10-14 days, until differentiation and budding;

The differentiation-promoting culture: the resistant buds screened in the antibacterial culture were transferred to the medium C3: pH6.0, MediumBase+0.5mg/L trans-zeatin nucleoside+1.5mg/L3-indoleacetic acid+ 80mg/L Kanamycin + 200mg/L Timentin;

The culture medium for rooting culture is: pH6.0, MediumBase+2mg/LIBA+200mg/L Timentin.

The purpose of the present invention is, through the combination of systemic gene silencing and grafting technology, first make the rootstock acquire resistance to the invading virus, and then the rootstock transduces its virus resistance to the scion so that the scion variety grafted on the rootstock can acquire the virus resistance. The idea that the present invention is different from the prior art is: the gene silencing object of the RNA interference vector constructed for transforming rootstocks is the gene of the invading virus, not the gene of the plant itself.

In the present invention, tomato is used as rootstock and scion test material, and cucumber mosaic virus CMV, which has a wide range of hosts, is used as target virus for specific experimental verification. Experimental data show that scion tomato has obtained good CMV virus resistance. Based on the concept of the present invention, the guidance of the experimental method recorded in the description and the routine experimental skills possessed by those of ordinary skill in the art, those skilled in the art can apply the method of the present invention to many kinds of viruses that are easy to be infected by viruses and can be grafted. In the plants, this application is all within the scope of protection of the present invention. That is, the method of the present invention is not limited to the tomato described in the specific examples.

In the present invention, tomato is used as a test material, and Cucumber Mosaic Virus (CMV) is used as a target object to verify the concept of the present invention. That is, by selecting 5 CMV gene OPF fragments and a total of 6 ihpRNA plant expression vectors fused with 5 gene fragments, using Agrobacterium-mediated transformation of tomato to make the transformed plants produce siRNA, and inoculating CMV to screen out transgenes with high virus resistance plants. Then use the T0 or T1 generation of resistant plants as the rootstock, and graft non-transgenic tomatoes, and the siRNA in the rootstock is transferred to the scion to enhance the resistance of the scion to CMV. At present, no resistance gene to CMV virus has been found in cultivated tomato varieties, and there are only disease-resistant varieties in cultivated tomato varieties. It is difficult to obtain CMV-resistant tomato varieties by conventional hybridization methods. Therefore, the method of transgenic by RNA interference is an effective way to quickly obtain CMV-resistant materials. In recent years, around the breeding of genetically modified organisms, my country has achieved international standards in the study of safety management and safety evaluation of genetically modified organisms, and has made remarkable progress. However, some people have always had doubts about the impact of resistance selectable marker genes involved in genetic engineering on food safety. However, the present invention cleverly avoids this issue, because the systemic resistance acquired by the scion is derived from the silencing effect of the small molecule RNA in the rootstock. Therefore, in theory, the scion genome of grafted transgenic silent plants and the resulting female/male gametes will not contain resistance marker genes, which prevents the drift of antibiotic resistance genes.

The present invention comprises the following steps:

a. For the 5 genes of cucumber mosaic virus (CMV), by comparing the tomato EST database, select the fragments of the ORF interval of the 5 genes of CMV, and construct 5 fragments containing CMV-specific genes and 1 fusion of 5 gene fragments by PCR and overlapping PCR The ihpRNA plant silencing expression vector of the gene fragment;

b. Utilize the improved genetic transformation system, transform tomato plants through Agrobacterium-mediated transformation, obtain transgenic positive plants through detection, and propagate transgenic plants by cuttings (T0 generation);

c. By inoculating CMV virus, comparing the CMV resistance of transgenic strains with different vectors, and screening out transgenic strains with high virus resistance;

d. Use resistant cutting seedlings (T0 generation) or T1 generation plants as rootstocks, graft wild-type plants (non-transgenic plants), inoculate CMV virus, and detect grafted plants that have acquired virus resistance;

e. extract rootstock and scion total RNA, reverse to cDNA, and detect the expression of antibiotic resistance gene by RT-PCR.

Description of drawings

Figure 1: Genome structure features of cucumber mosaic virus (CMV)

Figure 2: The carrier construction scheme adopted in the present invention

Figure 3: Construction of 5 vectors containing CMV gene-specific fragments

M2K/15K: Marker2K/15K;

A: Use PstI/SalI digestion to verify the cloning vector pUCCRNAi-1A～CP, and the cut out fragment is the inverted repeat sequence constructed into the cloning vector;

B: Construct the inverted repeat sequence from pUCCRNAi-1A～CP into the expression vector to obtain the pCAMBIA2300-1A～CP vector;

C: 1-3: PstI/SalI digestion to verify the expression vector pCAMBIA2300-1A-CP, the small fragments excised are inverted repeat sequences constructed into the cloning vector.

Figure 4: Construction of a fusion vector containing 5 CMV gene fragments

A.1: Marker is 2K, using R1AF/R1AR and R2AF/R2AR primers to amplify fragments 1AF and 2AF respectively;

A.2: Use the R1AF/R2AR primers to amplify the fusion fragment 1A+2A using the mixture of 1AF and 2AF as a template;

B.1: Use R2BF/R～RCPF/R primers to amplify fragments 2BF, 3AF and CPF respectively;

B.2: Use R2BF/RCPR primers to amplify the fusion fragment 2B+3A+CP using the mixture of 2BF, 3AF and CPF as a template;

C.: CMV5 uses R1AF/RCPR primers to amplify the fusion fragment CMV5 with 1A+2A and 2B+3A+CP mixtures as templates;

D.: PstI, PstI/SalI digestion to detect pCAMBIA2300-CMV5 vector.

Figure 5: The improved tomato transgenic steps adopted by the present invention

Figure 6: PCR detection of transgenic tomato (batch detection of some lines)

M: Marker2K; +: Plasmid pCAMBIA2300-1A as a template; CK: Non-transgenic tomato MoneyMaker as a template 1A～CMV5-1～9: Indicates the strips amplified with Actin1PF/IntornR as primers and corresponding transgenic lines of different target vectors as templates bring

Figure 7: Southern hybridization detection of some transgenic tomato lines

M: Marker15K; +: Plasmid pCAMBIA2300-1A as a template (not digested completely); CK: Non-transgenic tomato MoneyMaker as a template; T01A～CMV5.1～2: Southern hybridization bands of different target vectors corresponding to transgenic lines

Figure 8: RT-PCR detection of transgenic tomato plants

M: Marker2K; +: Plasmid pCAMBIA2300-1A as template; CK: non-transgenic tomato MoneyMaker as template; T01A～CMV5.1～2: 1AF～CPF and R1AF (T0CMV5) as upstream primers, IntronR as downstream primers, respectively. Amplify the bands corresponding to transgenic lines with different target vectors

Figure 9: Transgenic tomato cutting propagation and CMV challenge to screen anti-virus T0 generation lines

CK: Non-transgenic tomato plant MoneyMaker; T01A～CP.1～2 and T0CMV5.1～5: Indicate the cutting seedlings of different transgenic lines, and the virus resistance was identified after inoculation with CMV virus. The lines in the figure are some of the selected high Anti-strain

Figure 10: Propagation of transgenic tomato T1 generation and CMV challenge to screen anti-virus T1 generation lines

CK: Non-transgenic tomato plant MoneyMaker; T11A.1～CMV5.1: Indicates the T1 generation of different transgenic lines, and the virus resistance was identified after inoculation with CMV virus

Figure 11: Grafting technique used in the present invention

Figure 12: RT-PCR detection of antibiotic resistance gene NptII after grafting

M: Marker2K; +: Plasmid pCAMBIA2300-1A as a template; CK: Non-transgenic tomato MoneyMaker as a template; T01A～CMV5.1～2: Indicates PCR amplification using NptIIF/R as primers and corresponding transgenic lines of different target vectors as templates The top panel of the band indicates that there is no expression of NptII gene in the scion

detailed description

Example 1. For the cucumber mosaic virus (CMV) gene, 6 ihpRNA plant silencing expression vectors were constructed by comparing the tomato EST database;

Cucumber mosaic virus (CMV) is a member of the genus Cucumovirus in the family Bromoviridae. CMV is a single-stranded positive-sense RNA virus. The genome is divided into RNA1, RNA2 and RNA3. The genome size is 3389nt, 3035nt and 2197nt, respectively, and they are respectively encapsulated in three virions. Among them, RNA3 contains subgenomic RNA4 with a total length of 1000nt. RNA1 encodes nucleic acid replicase; RNA2 encodes two proteins: RNA-dependent RNA polymerase (RNA-dependent RNApolymerase, RdRP) and 2b protein, RdRP is related to virus infection, 2b protein is similar to a viral motor protein, and it has been proved by experiments that 2b The protein can weaken the RNA-mediated silencing of viral genes; RNA3 encodes a motor protein 3A protein, whose specific function is unknown but related to the transmission of virus cells; RNA4 encodes the viral coat protein: CP protein. The specific features of the CMV genome are shown in Figure 1.

The present invention aims at the 5 genes of cucumber mosaic virus (CMV), by comparing the tomato EST database, selecting the fragments of the ORF interval of the 5 genes of CMV (as shown in SeqIDNo.1, 2, 3, 4, 5), and by PCR and overlapping PCR constructs 5 ihpRNA plant silencing expression vectors containing CMV-specific gene fragments and 1 fusion of 5 gene fragments. The specific steps are as follows:

The primers used to amplify the 5 fragments respectively, the underlined part is the introduced restriction site:

1AF: 5'-ATT GTCGAC TGGATGCGTCCTGTGC-3'

1AR: 5'-ATC GGATCC TTGGGCGGACTTCTTG-3';

2AF: 5'-AAT GTCGAC TGTCCAACGCCAACC-3'

2AR: 5'-AGG GGATCC TTCGTTGTACCTACTC-3'

2BF: 5'-ATT GTCGAC TGGCTCGTATGGTGGA-3'

2BR: 5'-GAG GGATCC GCGAACCAATCTGTAT-3';

3AF: 5'-ATA GTCGAC AGCCCTGAAGCCATTA-3'

3AR: 5'-AGA GGATCC AAAGACCCTTCAGCAT-3';

CPF: 5'-ATT GTCGAC CTTTGTAGGGAGTGA-3'

CPR: 5'-ATC GGATCC TTTACGGACTGTCA-3';

Overlapping PCR primers used to amplify fusion fragments:

R1AF: 5'-TAT GTCGAC TGTGCCCGAGGGTATTG-3'

R1AR: 5'-TCAGTAGCACGGTTTAAATTTACAGATCACGCATTCAC-3'

R2AF: 5'-GTGAATGCGTGATCTGTAAATTTAAACCGTGCTACTGA-3'

R2AR: 5'-TTCTTCGCCTCCACCATACGCTTGATGAAAAGAGTCGTCGT-3'

R2BF: 5'-ACGACGACTCTTTTCATCAAGCGTATGGTGGAGGCGAAGAA-3'

R2BR: 5'-TCCACTGATGCTGAAGGGTCTGATAGAACGGTAGGAAGCG-3'

R3AF: 5'-CGCTTCCTACCGTTTCTATCAGACCCTTCAGCATCAGTGGA-3'

R3AR: 5'-CGAGTTAATCCTTTGCCGAACACGGTCGTATTGCTTCCTT-3'

RCPF: 5'-AAGGAAGCAATACGACCGTGTTCGGCAAAGGATTAACTCG-3'

RCPR: 5'-ATC GGATCC ATCTATTACCCTAAAGCCAC-3'

Cloning Vector Detection Primers:

M13+: 5'-AGGGTTTTTCCCAGTCACG-3'

M13-: 5'-GTGTGAAATTGTTATCCGCTC-3'

Expression vector detection primers:

Actin1PF: 5'-CCTCAGCATTGTTCATCGGTAGTT-3'

IntronR: 5'-TGTGTCACTCAAAACCAGATAAAC-3'

Cloning vector: pUCCRNAi, described in (Luo, A., Qian, Q., Yin, H. et al. (2006) EUI1, encoding aputative cytochrome P450monooxygenase, regulates internodeelongation by modulating gibberellin responses in rice. PlantCellPhysiol.47, 181–191.), also preserved in our laboratory, since It can be issued to the public for experimental research within twenty years from the date of application.

Plant expression vector: pCAMBIA2300-Actin1-ocs, described in (Fang, J., Chai, C., Qian, Q., Li, C., Tang, J., Sun, L., Huang, Z., Guo, X., Sun, C. and Liu, M. 2008. Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in rice. The Plant Journal. 2, 177-189), which is also preserved in this laboratory and can be released to the public for experimental research within 20 years from the date of application.

In the present invention, two groups of homologous enzyme cutting sites XhoI/SalI and BglII/BamHI are used in pUCCRNAi to select and amplify fragments of CMV5 gene ORF intervals such as SeqIDNo.1, 2, 3, 4, 5 The forward and reverse sequences of the fragments shown are respectively constructed into two sets of restriction sites of pUCCRNAi, that is, the forward and reverse sequences of each fragment are respectively constructed into two sets of restriction sites to obtain RNAi with inverted repeat sequences Cloning vector pUCCRNAi-1A, 2A, 2B, 3A, CP; through overlapping PCR, some fragments in the 5 fragments shown in SeqIDNo.1, 2, 3, 4, 5 were connected together to obtain the fragment shown in SeqIDNo.6 , the forward and reverse sequences of the fragment shown in SeqID No.6 were constructed into the two sets of restriction sites of pUCCRNAi to obtain the RNAi cloning vector pUCCRNAi-CMV5, and the primers were the above overlapping PCR primers.

The inverted repeat regions of the cloning vectors pUCCRNAi-1A, 2A, 2B, 3A, CP, and CMV5 were digested with endonuclease SalI/PstI and then connected to the plant expression vector pCAMBIA2300-Actin1-ocs. The vector construction scheme is shown in Figure 2 As shown, the plant expression vectors pCAMBIA2300-1A, 2A, 2B, 3A, CP, and CMV5 with ihpRNA were digested, and the digestion verification was shown in Figures 3 and 4. Sequencing results also showed that the construction positions of the selected forward and reverse sequences were correct.

The constructed plant expression vector was transformed into Escherichia coli competent cells, expanded and cultivated, and the plasmid was extracted for Agrobacterium transformation. The operation is as follows:

Step 1. Digestion and recovery of pUCCRNAi vector fragments

Extract the pUCCRNAi plasmid (Plasmid DNA mini-extraction kit) from Escherichia coli. Take 5ul of plasmid, use XhoI and BagII endonucleases, use 50ul enzyme digestion system, digest overnight at 37°C, and recover the plasmid fragment with a length of about 3K in the digested product; use DNA/RNA UV spectroscopy to detect agarose gel electrophoresis analysis Determine the concentration of recovered fragments. The recovered fragment was labeled as XhoI/BagII-pUCCRNAi.

Step 2. CMV gene PCR fragment digestion and recovery

Using primers 1AF/R～CPF/R, on the invasive cloning vector containing the whole CMV genome, obtained 5 CMV gene fragments respectively by the following PCR method, which were recorded as 1A, 2A, 2B, 3A and CP; verify its accuracy;

Dilution of primers: add deionized water directly to the synthesized primers to make a final concentration of 10umol/L.

Add the following ingredients in sequence to the PCR tube 50ul system 25ul system 10×PCR Buffer with Mg2+ 5ul 2.5ul template DNA 2ul 0.4-1ul 10mM dNTP (mixed) 4ul 2ul 10uM Primer F/Primer R 2ul 1ul Taq enzyme 1ul 0.5ul ddH2O Make up to 50ul Make up to 25ul

(2) After mixing, centrifuge slightly to the bottom of the tube, put the PCR tube into the PCR machine, and cover with a hot lid. Preheat the heated lid before amplification.

(3) The following amplification conditions

Pre-denaturation at 94°C for 3min, denaturation at 94°C for 30sec; annealing at 50-68°C for 30sec; extension at 72°C for 1min (1 minute for target fragments larger than 500bp, 40 seconds for target fragments smaller than 500bp), 35 cycles, 10min at 72°C, stored at 4°C

The PCR products were purified and recovered to obtain 5 CMV gene fragments. Take 20ul of the recovered fragments, use SalI and BamHI endonucleases, use 50ul enzyme digestion system, digest at 37°C for 6h, and recover the plasmid fragments corresponding to the sequence length shown in SeqIDNo.1-5 in the digested products. Take 1 ul of recovered fragments and perform gel electrophoresis to judge the recovered quality. CMV recovered fragments were labeled SalI/BamHI-1A, SalI/BamHI-2A, SalI/BamHI-2B, SalI/BamHI-3A, SalI/BamHI-CP.

Step 3. Ligation and transformation of pUCCRNAi-1A-CPF into Escherichia coli

Take 4ulXhoI/BagII-pUCCRNAi and 8ulSalI/BamHI-1A, SalI/BamHI-2A, SalI/BamHI-2B, SalI/BamHI-3A, SalI/BamHI-CP to connect respectively:

reaction system reaction system 10×T4DNA Ligation Buffer 2ul 5ul T4DNA Ligase (1U/ul) 2ul 5ul PCR fragment after enzyme digestion and recovery 8ul 20ul Carrier (50-400ng) 4ul 10ul sterile deionized water Make up to 20ul Make up to 50ul

Flick, mix, and centrifuge briefly. The connection product was obtained by reacting overnight at 16°C for 20 hours. Inactivate at 65°C for 10 minutes.

Take 10ul of the ligation product, and transform Escherichia coli competent according to the method (8. Transformation of Escherichia coli plasmid DNA) (competent Escherichia coli Trans5aChemicallyCompetentCell was purchased from Beijing Quanshijin Biotechnology Co., Ltd.)

(1) Add 3ul of 1-10ug of plasmid DNA to each tube of prepared competent E. coli (100ul/tube), (the ligation product does not exceed 1/10 of the competent) flick and mix well.

(2) Place the mixture in an ice-water bath for 30 minutes and mix well by flicking.

(3) Heat shock in a water bath at 42°C for 60 sec, then quickly transfer to ice for 2 min, be careful not to shake the centrifuge tube.

(4) Add 500ul LB liquid medium in a sterile 37°C warm bath on the ultra-clean workbench, and mix well.

(5) Pre-cultivate for 1 hour at 37°C with shaking at 200 rpm to obtain plasmid DNA transformed bacterial liquid. (Plates with antibiotics prepared during the period)

(6) Centrifuge at 4000rpm for 5 minutes to remove 400ul of the supernatant, and take 50ul of the rest to spread on a plate for screening. When spreading the plate, use ampicillin (Amp) at a final concentration of 50mg/L as the antibiotic to select positive transformants.

(7) Pick a positive single colony with a 20ul pipette tip, add it to 1ml LB liquid medium containing Amp, and incubate on a shaker at 250rpm at 37°C for 6h; take 2ul of the bacterial liquid, and follow the PCR method and system in step 2 (using the cloning carrier to detect primers M13+/- detected transformants, the positive result was the length of the CMV gene fragment plus 360bp, and PCR was selected as a positive transformant, which was verified by sequencing; the obtained Escherichia coli positive bacteria were labeled as pUCCRNAi-1AF, pUCCRNAi-2AF, pUCCRNAi-2BF respectively , pUCCRNAi-3AF, pUCCRNAi-CPF.

Step 4. Digestion and recovery of pUCCRNAi-1A-CPF vector fragments

The plasmids pUCCRNAi-1AF, pUCCRNAi-2AF, pUCCRNAi-2BF, pUCCRNAi-3AF and pUCCRNAi-CPF were extracted from Escherichia coli respectively.

Take 5ul plasmids respectively, use SalI and BamHI endonucleases, use 50ul enzyme digestion system, digest overnight at 37°C, and recover plasmid fragments with a length of about 3K in the digested products; take 1ul recovered fragments, and use the method DNA/RNA UV spectroscopy Detection and agarose gel electrophoresis analysis to determine the concentration of recovered fragments. The five recovered fragments were labeled as SalI/BamHI-pUCCRNAi1AF, SalI/BamHI-pUCCRNAi2AF, SalI/BamHI-pUCCRNAi2BF, SalI/BamHI-pUCCRNAi3AF, SalI/BamHI-pUCCRNAiCPF, respectively.

Step 5. Ligation and transformation of pUCCRNAi-1A-CP into Escherichia coli

According to the method of connecting the carrier and gene fragments in step 3, take the fragments recovered by enzyme digestion of the vector obtained in step 4 and the 5 kinds of CMV genes obtained in step 2, and press the combination

SalI/BamHI-pUCCRNAi1AF+SalI/BamHI-1A,

SalI/BamHI-pUCCRNAi2AF+SalI/BamHI-2A,

SalI/BamHI-pUCCRNAi2BF+SalI/BamHI-2B,

SalI/BamHI-pUCCRNAi3AF+SalI/BamHI-3A,

SalI/BamHI-pUCCRNAiCPF+SalI/BamHI-CP, for connection, after adding other components, use 20ul system, connect overnight at 16°C;

Take 10ul of the ligation product, transform Escherichia coli competent (the method is the same as step 3), take 2ul of the positive bacterial liquid and use the 50ul system according to the method, and use the cloning vector detection primer (M13+/-) to detect the positive result of the transformant by PCR (positive result is 2× The length of the CMV gene fragment plus 360bp); PCR-positive transformants were selected and verified by sequencing; the transformants with positive verification results were marked as pUCCRNAi-1A, pUCCRNAi-2A, pUCCRNAi-2B, pUCCRNAi-3A, pUCCRNAi-CP respectively.

Step 6. Digestion and recovery of pCAMBIA2300-Actin1-ocs vector fragment

The pCAMBIA2300-Actin1-ocs plasmid was extracted from Escherichia coli. Take 5ul of the plasmid, use SalI and PstI endonucleases, use 50ul enzyme digestion system, digest at 37°C overnight, and recover the plasmid fragment of about 10Kb in length in the digested product; take 1ul of the recovered fragment, and use DNA/RNA UV spectroscopic detection And agarose gel electrophoresis analysis to determine the concentration of recovered fragments.

The recovered fragment was labeled SalI/PstI-pCAMBIA2300.

Step 7.1A ~ CPihpRNA fragment digestion and recovery

Extract the pUCCRNAi-1A, pUCCRNAi-2A, pUCCRNAi-2B, pUCCRNAi-3A, pUCCRNAi-CP plasmids from the positive transformants in step 5. Take 5ul of the plasmid, use SalI and PstI endonucleases, use 50ul enzyme digestion system, digest overnight at 37°C, and recover a small fragment of about 700bp in the digested product (the ihpRNA fragment contains the forward and reverse CMV gene-specific fragment plus the inner sub-fragment); take 1 ul of the recovered fragment, and use DNA/RNA UV spectroscopic detection agarose gel electrophoresis analysis to determine the concentration of the recovered fragment. The five recovered fragments were labeled as SalI/PstI-1A, SalI/PstI-2A, SalI/PstI-2B, SalI/PstI-3A, SalI/PstI-CP, respectively.

Step 8. Ligation and transformation of pCAMBIA2300-1A～CP into Escherichia coli

Take 4ul of SalI/PstI-pCAMBIA2300 obtained in step 6 and 8ul of SalI/PstI-1A, SalI/PstI-2A, SalI/PstI-2B, SalI/PstI-3A, and SalI/PstI-CP obtained in step 7 for carrier and The connection of gene fragments, the method is the same as step 3.

Take 10ul of the ligation product respectively, and transform Escherichia coli into a competent state, and the operation is the same as that described in step 3;

Take 2ul of the transformed bacteria liquid, use the 50ul system, and use the expression vector detection primer Actin1PF/IntronR (the upstream primer Actin1PF matches the expression vector Actin1 promoter region, and the downstream primer IntronR matches the Intron in the ihpRNA structure) for PCR detection of positive transformants, ( A positive result is the length of the CMV gene fragment plus about 323bp);

Select PCR-positive transformants, add them to 50ml LB liquid medium containing Amp at a volume ratio of 1:1000, and culture them on a shaking table at 250rpm at 37°C for 12h; extract plasmids, and the resulting plasmids are respectively labeled as pCAMBIA2300-1A, pCAMBIA2300-2A, and pCAMBIA2300-2B , pCAMBIA2300-3A, pCAMBIA2300-CP.

Step 9. Construction of pCAMBIA2300-CMV5 vector

Using pUCCRNAi-1A and pUCCRNAi-2A plasmids as templates, primers R1AF/R1AR and R2AF/R2AR were used to amplify fragments 1AF and 2AF, respectively, and the PCR products were recovered to obtain purified fragments 1AF and 2AF; an equal mixture of fragments 1AF and 2AF was used as As a template, fragment 1A+2A is amplified by PCR using R1AF/R2AR primers, and the PCR product is recovered to obtain purified fragment 1A+2A.

Using pUCCRNAi-2B, pUCCRNAi-3A and pUCCRNAi-CP plasmids as templates, primers R2BF/R2BR, R3AF/R3AR, RCPF/RCPR were used to amplify fragments 2BF, 3AF and CPF, and the PCR products were recovered to obtain purified fragments 2BF, 3AF and CPF: Fragment 2B+3A+CP is amplified by PCR using an equal mixture of fragments 2BF, 3AF and CPF as a template, using R2BF/RCPR primers, recovered and purified.

Using the mixture of fragments 1A+2A and 2B+3A+CP as templates, the fragment CMV5 was amplified by PCR using R1AF/RCPR primers, recovered and purified.

Obtain pUCCRNAi-CMV5 according to the above 3 steps, digest pUCCRNAi-CMV5F according to step 4 to obtain SalI/BamHI-pUCCRNAiCMV5F, obtain the cloning vector pUCCRNAi-CMV5 according to step 5 and obtain SalI/PstI by the method of step 7-- CMV5, the SalI/PstI-pCAMBIA2300 in step 6 and the SalI/PstI--CMV5 obtained in step 7 were obtained according to the operation in step 8 to obtain the expression vector pCAMBIA2300-CMV5.

Example 2. Functional verification of RNA interference carrier

Material:

ZT (Trans-Zeatin-Riboside, trans-zeatin riboside), Tim (Timentin, Timentin), folic acid, As (acetobutanone), Kan (kanamycin), IAA (3-indole acetic acid) , IBA (3-indolebutyric acid), and MSsalt (M0404) were purchased from Sigma.

Tomato variety: MoneyMaker, which is susceptible to CMV.

Medium preparation: (unit: L)

(1) 1/2MS: pH5.81/2MSsalt+15g|sucrose+6.5g|agar

(2) Medium A: pH5.8 MS salt+30g | sucrose

(3) Medium B: pH5.8MS salt+30g|sucrose+6.5g|agar+1mg|ZT

(4) Medium Base: pH6.0MSsalt+20g|Sucrose+6.5g|Agar+100mg|Inositol+0.1mL|10000×Folic acid

(5) Medium C: pH6.0MediumBase+2mg|ZT+200mg|Tim+100mg|Kan

(6) Medium C1: pH6.0MediumBase+1mg|ZT+80mg|Kan+200mg|Tim

(7) Medium C3: pH6.0MediumBase+0.5mg|ZT+1.5mg|IAA+80mg|Kan+200mg|Tim

(8) Medium D: pH6.0MediumBase+2mg|IBA+200mg|Tim

method:

The six plant expression vectors pCAMBIA2300-1A, 2A, 2B, 3A, CP, CMV5 obtained and identified in Example 1, all target fragments SeqIDNo.1-6, and the tomato EST database http://solgenomics .net/ for comparison, all fragments have no continuous 20nt or more complete complementary regions with the tomato EST, which ensures that the siRNA formed by the RNAi vector in the transgenic plant will not target the important genes of the tomato plant.

Step 1. Preparation of transgenic plants

In the method of the present invention, the existing tomato genetic transformation system (Frary, A. and VanEck, J. 2005. Organogenesis From Transformed Tomato Explants. Transgenic plants: methods and protocols. 141-150.) is improved, and the transformation time is shortened. Its main steps are as follows: See (Figure 5):

(1) Sowing for 6-7 days: soak the seeds with sterile water for 5-8 hours; sterilize with 20% sodium hypochlorite for 10 minutes; wash with sterile water for 5 times, each time for about 5 minutes; accelerate germination for two days in a 30°C incubator; 1/2MS medium, and then transferred to a 26-28°C light incubator for about 5 days, and the cotyledons can begin to unfold. Improvement: Accelerating germination after soaking can shorten the germination by about 1 week, and the germination is neat.

(2) Pre-cultivation of cut cotyledons for 2 days: Before the true leaves appear, when the cotyledons have just emerged from the seed coat and are not fully unfolded, cut off both ends of the cotyledons by 1-2 mm, and then cut across the middle, and cut each cotyledon into two Collect the pieces in the culture dish containing medium A, about 50-80 pieces each time. Pour off the culture medium A, use a scalpel to carefully remove the leaf disk (do not pinch) on the sterile filter paper, blot dry, then transfer to the culture medium B covered with filter paper, with the cotyledon leaves facing up, pre-cultivate in low light for 2 days. Improvement: the differentiation efficiency of the cotyledon collected in the period described in the present invention is much higher than that after the emergence of true leaves.

(3) Preparation of engineering bacteria:

a. Adjust the electric shock meter so that the electric pulse is 25uF, the voltage is 2.5KV, and the resistance is 400Ω.

b. Take out the Agrobacterium EHA105 competent cells from the -70°C refrigerator, and place them on ice to thaw.

c. Add 1ul plasmid to 50ul EHA105 competent cells, and mix gently.

d. Add the above mixed suspension to the bottom of the electric shock cup, and quickly put the electric shock cup into the electric shock instrument.

e. According to the parameters set in a, start the electric pulse to the cell. After the electric shock, take out the sample as soon as possible, and add 1 ml of YEB-free medium.

f. Transfer the bacterial solution obtained in e into a 1.5ml centrifuge tube, and shake at 200 rpm at 28°C for 3 hours.

g. Take 50-100ul bacterial liquid and spread it on the YEB medium added with antibiotics Kan and rifampicin (Rif), culture at 28°C for 2-3 days, until colonies grow out.

Note: All operations are best performed on ice, and the electric shock cup is pre-cooled on ice before electric shock.

h. Melt the LB solid medium in a microwave oven, cool it down to about 50°C, and add the corresponding antibiotic (usually Amp) on the ultra-clean workbench.

i. Add 16ul of IPTG (50mg/ml) and 40ul of X-gal (20mg/ml) on the surface of the plate after plating 20ml/block, spread evenly with a sterile elbow glass rod, and place it on a clean bench in the dark For 1 hour, the dimethylamide dissolved in X-gal was volatilized as much as possible.

j. Add the transformed bacteria solution on the plate and spread it evenly with a sterile elbow glass rod (the remaining bacteria solution should be stored at 4°C).

k. Place the plate forward for 1 hour to allow the bacterial solution to absorb. Then put it upside down into the incubator, and cultivate at 37°C for 12-16h until clear colonies appear.

l. After the blue and white spots appear, place them in a refrigerator at 4°C to allow them to fully develop their color (the white spots may be positive clones, pick them out for expanded culture, and then do colony PCR or plasmid PCR/enzyme digestion detection).

(4) Engineering bacteria infection and co-cultivation for 2 days: The engineering bacteria were streaked 2 days in advance, and then the single bacteria were shaken in the dark at 26°C for about 16 hours to OD600=0.6-0.8. Centrifuge at 5000 rpm for 5 minutes to collect the bacterial liquid, and use the culture medium A was adjusted to about OD600=0.5, and the final concentration of 300uM AS was added. Infect the pre-cultured leaf disc explants for 10 minutes, blot them dry with sterile filter paper, place the leaf back up (do not pinch) on medium B covered with filter paper, and culture in the dark at 26°C for 2 days.

(5) Bacteriostasis screening culture for 7-10 days: transfer to medium C, culture with the leaf back facing up in the light; transfer once every 10 days, under normal circumstances, green calluses or buds will appear in about 2 weeks. Wait for buds to appear on the callus. Then transfer the explants with buds to C1 and culture them for 10-14 days, and they will gradually differentiate and sprout in 2-3 weeks. Improvement: High concentration of ZT (2mg/L) is good for division, but too fast division may cause negative regenerated shoots to escape antibiotics. In the C1 medium of the present invention, reduce ZT in time and use a higher Kan concentration (100mg/L) in C prevents this.

(6) Differentiation-promoting culture for 10-20 days: the regenerated shoots grow about 0.5 cm. Parts of the albino explants were excised and the resistant shoots were transferred to fresh C3. Part of the ungerminated leaf discs will be albino, and the cut end will be browned without signs of healing. Such leaf discs should be removed in time. For leaf discs with the ability to grow regenerated buds, small groups of calluses will generally appear on the incision, and then differentiate and sprout. Improvement: Long-term exposure to ZT tends to form deformed buds. The improved medium C3 of the present invention contains low concentration of ZT and adds auxin IAA, which can effectively promote the differentiation and growth of normal buds.

(7) Rooting culture for 15 to 30 days: After the resistant bud stem grows to 1cm, remove the callus, cut it off and put it in medium D for rooting culture. Improvement: the antibiotic Kan has a significant effect on plant rooting, and the present invention does not add the antibiotic Kan to the rooting medium, which can effectively increase the rooting rate.

(8) Field cultivation: Slowly open the bottle cap 3 days before transplanting, transfer to sterilized culture soil and wrap it in a transparent plastic bag. After 1 week, gradually uncover the plastic bag and transplant to the field. The leaves to be tested were wiped with sodium hypochlorite 2 days before the PCR test, and the leaves were washed with sterile water before subsequent testing.

The above-mentioned operating method of the present invention compares with the effect of existing commonly used method:

(1): Germination after soaking can shorten the germination by about 1 week, and the germination is neat.

(2): The differentiation efficiency of the cotyledons collected during the period described in the present invention is much higher than that after the emergence of true leaves.

The cotyledons collected during the period described in the present invention, that is, "before the true leaves appear, when the cotyledons have just emerged from the seed coat and are not fully expanded", the differentiation efficiency reaches 100% (number of differentiated buds/number of leaf disks) and each leaf disk More than 2 buds can be differentiated; 2 days after the emergence of true leaves, the survey differentiation efficiency is 50%; 5 days after the appearance of true leaves, the survey differentiation efficiency is less than 10%.

(3): The timely reduction of ZT in the C1 medium of the present invention and the use of a higher Kan concentration in C can effectively prevent false positives.

The average false positive rate (number of false positive buds/number of differentiated buds) obtained by the method of the invention is 24%; if 2mgZT is used, the false positive rate is 40%. Note: The concentration of Kan used in the present invention is suitable for MoneyMaker varieties, while we have tested tomato varieties Ponceau and mic-Tom, and the suitable concentrations are 50mg/L and 75mg/L respectively.

(4): The improved medium C3 of the present invention contains a low concentration of ZT and increases the auxin IAA, which can effectively promote the differentiation and growth of normal buds.

The deformity rate of resistant buds (number of deformed buds/number of resistant buds) in the method of the present invention is 5%, while the deformity rate of buds that are not added with IAA and are kept in 1 mg/LZT is as high as 22%.

(5): Kan's effect on rooting.

The present invention has tested the influence of different concentrations on rooting. Since the number of transgenic positive buds is limited, the selected buds are buds differentiated from uninfected cotyledons, 20 each. 100mg/L concentration Kan plant rooting rate (number of rooted plants/bud number after 2 weeks) 10% (2 plants); 75mg/L concentration Kan plant rooting rate 40% (8 plants); 50mg/L concentration Kan plant rooting 60% (12 strains); 0mg/L concentration can see the rooting rate of 90% (18 strains). After 2 weeks to 1 month, under the concentration of 100mg/L Kan, the unrooted plants did not take root all the time, and the leaves turned yellow or turned white; under the concentrations of 75mg/L and 50mg/L Kan, 3 and 2 long roots were increased respectively Roots also appeared in the remaining 2 strains at a concentration of 0 mg/L.

Step 2. Rapid PCR detection.

Use the GenStar Rapid Plant PCR Kit and instructions for PCR detection of transgenic plants, the primers used are:

Actin1PF: 5'CCTCAGCATTGTTCATCGGTAGTT3',

IntronR: 5'TGTGTCACTCAAAACCAGATAAAC3', and

NptIIF: 5'GATACCGTAAAGCACGAGGAA3'

NptIIR: 5'TGACTGGGCACAACAGACAAT3'

Use specific primers to detect the virus gene in the resistant plants by PCR.

Use NptIIF and NptIIR as primers to detect the NptII resistance marker gene, and the amplified target fragment should be a 673bp fragment.

Design the upstream primer Actin1PF in the vector promoter region and the intron IntronR in the vector as the downstream primer, amplify the target fragment to detect the inserted fragment, and determine the positive transgenic plants according to the detection results shown in Figure 6.

Step 3. Southern hybridization analysis of transgenic plants

For the positive plants detected by PCR, the young leaves were taken, the total DNA was extracted by CTAB method, digested with EcoRI, and analyzed by Southern hybridization to further confirm the integration of the exogenous target fragment in different transgenic plants. The operation is as follows:

Probe labeling:

Using the DNA of the positive plants detected by PCR as a template, the dUTP (Roche) mixture was used for secondary PCR amplification to obtain dUTP digoxin-labeled probes.

Mass genome digestion:

(1) Enzyme digestion system:

Genomic DNA60ug about 100ul

10×BufferH60ul

Endonuclease (EcoRI) 30ul

Final volume 600ul

(2) Incubate overnight at 37°C; take 5ul of the digested product for electrophoresis analysis to detect the digesting effect;

(3) After complete digestion, add 0.1 times (60ul) volume of 3mol/L NaAc and 2 times (1200ul) volume of absolute ethanol (-20°C), mix well and place at -20°C for 2 hours;

(4) Centrifuge at 12000rpm at 4°C for 10min, carefully discard the supernatant; add 1ml of 75% ethanol to wash once, blow dry the precipitate in an ultra-clean bench, dissolve in 30ulddH2O for later use.

(5) Prepare 1% agarose gel with 0.5×TBE, the thickness of the gel is about 0.5cm; add the test sample: 30ul (about 10ug) DNA digestion concentrate solution plus 6×bromophenol blue solution; 120V voltage ( About 6V/cm) electrophoresis for 5min, after the bromophenol blue enters the gel, reduce the voltage to 100V (about 4V/cm), electrophoresis for 2h.

DNA denaturation:

(1) Bromophenol blue migrates to about 8-10cm (3/4) away from the gel hole to stop electrophoresis, take out the gel block from the gel tank, cut off the excess part of the gel block outside the swimming lane, and accurately measure the length and width of the gel. Cut off a corner of the gel as a mark;

(2) Glue is placed in a large Petri dish, soaked in Denaturation solution, and gently shaken on a shaker for 15min x 2 times to denature the DNA;

(3) Wash the gel slightly with ddH2O, put it into the Nenutralization solution and submerge the gel for 15min x 2 times (without shaking). Transfer film/Fixed film:

(1) Use whatman abrasive tool (TurboBlotter ^TM transfer device), stack as required, put 4 layers of large absorbent paper, then put 5 layers of 4*7cm absorbent paper, put 2 layers of smooth absorbent paper on the top layer; above the smooth absorbent paper Put a nylon membrane slightly larger than the lower absorbent paper (2*SSC pre-soaked for 5min) and carefully drive away the air bubbles between the nylon membrane and the smooth absorbent paper with a glass rod; take out the gel, and carefully protrude the gel hole with a blade The gel is cut flat, and the gel hole is placed in the middle of the absorbent cloth. Use a glass rod to drive away the air bubbles between the gel and the filter paper; cut a strip of salt bridge paper 3 times the width of the gel, cover it on the gel, fold both ends to communicate with the 20×SSC in the mold groove, and cover it with an absorbent paper ;

(2) After transferring the membrane for 4 hours, take out the nylon membrane, put it on a piece of filter paper, and use a pencil to mark the position of the sampling hole and the sample number on the nylon membrane;

(3) Take out the nylon membrane, put the DNA-bound side up on a piece of filter paper soaked in 10×SSC, and crosslink twice with ultraviolet crosslinking 1200*100uj/cm2.

Pre-hybridization, hybridization:

(1) Pre-hybridization: put the nylon membrane into the hybridization tube, add an appropriate amount of 30ml hybridization solution preheated at 42°C, and pre-hybridize in the hybridization oven at 42°C for 30 minutes;

(2) Probe denaturation: take an appropriate amount of probe (approximately 25ng of probe is added to 1ml of hybridization solution), add ddH2O to 100ul, 95°C for 5min, and quickly place on ice;

(3) Add the denatured probe to the hybridization solution preheated at 42°C, and mix gently to prevent bubbles from darkening the background;

(4) Pour out the pre-hybridization solution, add the hybridization solution, shake gently, and hybridize at 41°C (NptII) for 4 hours or overnight.

Membrane washing and color detection:

(1) Take out the hybridized membrane and put it into a clean plastic box of suitable size, rinse twice with low stringency washing solution at room temperature (shake gently when rinsing), 5 minutes each time;

(2) Rinse twice at 68°C with 68°C preheated high-rigority washing solution for 15 minutes each time;

(3) Gently rinse with Washingbuffer for 3 minutes;

(4) Add 50ml 1×blockingsolution and incubate for 30min at room temperature;

(5) Add 20ml Anti-bodysolution and react at room temperature for 30 minutes;

(6) Wash the membrane twice with Washingbuffer, 15min each time;

(7) Add 20ml Detectionbuffer to balance for 2-5min;

(8) Put the membrane in a clean petri dish, add an appropriate amount of Colorsubstratesolution, and develop the color in the dark for 16 hours without shaking; pour off the color developing solution, add TEbuffer and rinse for 5 minutes to stop the color developing reaction.

Main reagents and preparation:

(1) 20×SSC: Weigh 175.3g NaCl and 88.2g sodium citrate, dissolve in 800ml distilled water, adjust the pH to 7.0 with HCl, dilute to 1L with distilled water, autoclave, and store at room temperature;

(2) 1MTris-HCl (pH8.0): Weigh 12.114g Tris-base, dissolve in 80ml distilled water, adjust the pH to 8.0 with HCl, dilute to 100ml with distilled water, autoclave, and store at room temperature;

(3) 0.5MEDTA (pH8.0): Weigh 18.61g of EDTA, dissolve in 80ml of distilled water, adjust the pH to 8.0 with NaOH, dilute to 100ml with distilled water, autoclave, and store at room temperature;

(4) TEBuffer: Take 5ml of 1MTris-HCl (pH8.0), 1ml of 0.5MEDTA (pH8.0), dilute to 500ml with distilled water, adjust the pH to 8.0, autoclave, and store at room temperature;

(5) 10% SDS: Weigh 10g of SDS, dissolve in 90ml of distilled water, heat to 68°C to dissolve, adjust the pH to 7.2 with hydrochloric acid, and dilute to 100ml;

(6) Maleic Acid Buffer: Weigh 11.067g of maleic acid and 8.766g of NaCl and dissolve in 800ml of distilled water, adjust the pH to 7.5 with NaOH (solid), adjust the volume to 1L with distilled water, autoclave, and store at room temperature;

(7) WashingBuffer: Add 0.3% (v/v) Tween 20 to MaleicAcidBuffer after sterilization;

(8) DetectionBuffer: Weigh 1.1688g NaCl and dissolve it in 100ml distilled water, add 20ml 1MTris-HCl (pH8.0), adjust the pH to 9.5 with NaOH, adjust the volume to 200ml with distilled water, autoclave, and store at room temperature;

(9) Denaturation solution Ⅰ (Denaturation solution): 16gNaOH, 58.43gNaCl, dilute to 1L, autoclave, store at room temperature;

(10) Denaturing solution II (Nenutralization solution): 58.43g NaCl, 0.5MTris-HCl (pH7.2) 500ml, dilute to 1L, autoclave, store at room temperature.

(11) Low stringency washing solution: Dilute 20×SSC with distilled water to 2×SSC, and add 10% SDS at a ratio of 100:1 (2×SSC: 10%SDS) to make 2×SSC/0.1%SDS;

(12) High-rigority washing solution: dilute 20×SSC with distilled water to 0.5×SSC, and add 10% SDS at a ratio of 100:1 (2×SSC: 10%SDS) to make 0.5×SSC/0.1%SDS;

(13) BlockingSolution: Dilute 10×Blockingsolution 10 times with Maleic Acid Buffer (freshly prepared);

(14) AntibodySolution: Centrifuge Anti-Digoxigenin-AP10000rpm for 5min, and dilute it with BlockingSolution at 1:5000;

(15) Colorsubstratesolution: Dilute NBT/BCIP at 1:50 (200ul: 10ml) with DetectionBuffer, and store in the dark;

A total of 63 PCR-positive transformed plants (among them 1A9, 2A11, 2B12, 3A8, CP13, and CMV510) were obtained in the present invention. Part of the results of southern hybridization are shown in Figure 7, and it was found that most of the positive plants existed in the form of multiple copies.

Step 4. RT-PCR detection of siRNA precursor in transgenic plants

For the tomato transgenic lines that were positive in the molecular detection in step 2, RNA was extracted, and cDNA was synthesized by reverse transcription. Using 1AF～CMV5 and IntronR as primers, RT-PCR was used to detect the expression of siRNA precursors. The results are shown in Figure 8, showing that each positive The precursors of the strains all express, which proves that the constructed plant expression vector has been successfully transformed into tomato and obtained expression.

Example 3. Cutting Propagation of Transgenic Plants The resistance of each target gene transgenic line was determined.

Since the T0 generation of transgenic positive plants needs to be preserved after being transferred from the sterile room to the field for survival, it is not suitable for direct virus inoculation. Therefore the present invention adopts the mode of cuttage to carry out vegetative propagation, can solve this problem. At the same time, due to vegetative propagation, cutting propagation can provide more rootstock materials with the same genetic background for subsequent grafting.

Step 1. When the plant grows to a certain height, the mother plant is used to obtain seeds, take the lateral buds grown from the leaf axils, or take the stem section with a knot, collect the lateral buds or stem sections in a clean triangular flask, add 0.2 mg/LIBA sterile water 50ml, after 1 to 2 weeks, new roots will emerge from the bottom of the lateral buds (stems later). The rooted plants were transferred to the sterilized medium and placed in the greenhouse for cultivation. The cutting seedlings were all positive in rapid PCR detection.

When the four leaves of the cutting seedlings are unfolded, they are used for subsequent virus inoculation detection.

Step 2. Determination of the inoculation system of cutting seedling CMV virus

The cutting seedlings (compared to MoneyMaker; MM) were inoculated with the virus according to the following national standard operation method. After 21 days, many cutting seedlings had no obvious symptoms, and typical symptoms appeared only after 50-60 days. It is believed that this is because the cutting seedlings have passed the childhood stage and entered the mature stage compared with the seedlings, and their disease resistance has been improved to a certain extent. In order to shorten the identification time and obtain reliable disease resistance results, the present invention changed the rubbing inoculation (twice) to three consecutive times (with an interval of 1 week), and observed that all control cuttings showed symptoms in about 30 days. Therefore, it was determined that after the cutting seedlings were isolated and survived from the mother plant, the CMV virus was inoculated after the 4 leaves of the cutting plants were unfolded, and the inoculation was repeated every other week, a total of 3 times, and the CMV virus resistance was investigated after 30 years of growth.

Steps:

(1) Prepare 0.03mol/L, pH8.0 phosphate buffer. A: 0.03mol/L disodium hydrogen phosphate solution: Weigh 10.74g Na2HPO4 and dissolve in 1000mL water, shake well. B: 0.03mol/L Potassium Dihydrogen Phosphate Solution: Weigh 4.08g of Potassium Dihydrogen Phosphate, dissolve it in 1000mL of water and shake well. Prepare liquid A with liquid B to have a pH of 8.0.

(2) Preparation of the virus: Tomato CMV virus was propagated on the common tobacco "Blade Sansheng". The virus was collected and weighed after the onset of the virus, and the phosphate buffer was added at a ratio of 1:5, smashed, and immediately filtered through double-layer gauze. use.

(3) Inoculation method: The first inoculation is carried out when the tomato grows to 2 true leaves, and friction inoculation (use a duster to sprinkle emery (600 mesh) on the surface of the plant to be inoculated; use the friction inoculation method, that is, fully wash Dip the inoculation suspension with a clean finger, rub it slightly on the leaf surface to cause slight injuries, inoculate the first to second true leaves of each plant, and rinse the excess virus juice on the leaf surface with clean water immediately after inoculation. Inoculate twice, with an interval of 3 days ~5d.The inoculation is carried out in the greenhouse, the indoor temperature on the day of inoculation is 26°C-30°C, after that, the daytime temperature should be controlled at 24°C-30°C as much as possible, and the nighttime should not be lower than 20°C, with regular light and normal cultivation management.

(4) Disease investigation and grading standards

Table 1 Classification of disease severity of tomato virus disease caused by cucumber mosaic virus

symptom description 0 Asymptomatic. 1 The veins of the heart leaves are bright veins, and 1 to 2 true leaves present mosaic leaves. 3 Middle and upper leaves mosaic. 5 Most leaves are mosaic, and a few leaves are deformed or obviously shrunken. 7 Most of the leaves are heavily mosaic, some of the leaves are deformed and slender, and the plants are obviously dwarfed.

9 Almost all leaves heavy mosaic, most leaves misshapen, fern leaf. Plants are severely dwarfed or even dead.

(5) Survey time: about 21 days after inoculation.

(6) Disease record: investigate and record the disease level of each identified plant, and calculate the disease index. The disease index is calculated according to the following formula:

$\mathrm{<span\; class="notranslate">\; DI</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; the\; s</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; no</span>}<span\; class="notranslate">\; )</span>}{\mathrm{<span\; class="notranslate">\; N</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; S</span>}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}$

In the formula: DI - disease index;

∑——the sum of the product of the representative value of each disease level and the corresponding number of plants of each disease level;

s——the representative value of each disease level;

n——the number of plants of each disease level;

N - the total number of plants under investigation;

S——The representative value of the highest disease level.

(7) Resistance evaluation: When the susceptible control material reaches its corresponding susceptibility level (DI≥50), the identification of this batch is considered valid. The resistance level is determined based on the average value of the disease index of the identified materials for 3 repetitions, and the classification criteria are shown in the table below.

Evaluation Standards of Tomato Resistance to Cucumber Mosaic Virus

Disease Index (DI) Resistance evaluation DI=0 Immune (I) 0<DI<10 Highly resistant (HR) 10≤DI＜30 Disease Resistant (R) 30≤DI<50 Moderately resistant (MR) 50≤DI＜70 Susceptible (S) 70≤DI≤100 Highly sensitive (HS)

Step 3. Identification of transgenic line cutting seedling virus resistance

After the virus was inoculated according to the above method, the virus resistance of the T0 generation transgenic tomato was identified. Specifically, about 10 transgenic mother plants of each T0 generation were propagated by cuttings, and the resistance results were counted after the virus was inoculated. The results are shown in the following table (Figure 9):

It can be seen from the results in the table that all transgenic lines have a high degree of CMV virus resistance, most of which are highly resistant plants, and CMV5 is not obvious compared with other lines, the possible reason is that the investigated transgenic lines have high There are many resistant strains, but their advantages have not been reflected.

Note: The vast majority of high-resistant plants are immune types (ie asymptomatic) according to the classification formula, but we have detected CMV virus in some duplicate strains (cutting strains) of these plants, but the content is very low. Given that RNAi is The phenomenon of gene silencing at the post-transcriptional level, we believe that transgenic plants are different from immune plants in the strict sense, and it cannot guarantee that CMV will not infect plants completely. Therefore, we collectively classify such disease-resistant strains as high-resistance types.

The results proved that the use of transgene-generated RNA interference to fight against CMV virus is very effective. In order to facilitate later management, the lines were renumbered as T01A1~9 according to the resistance (T0 means T0 generation, 1A means the target gene corresponding to the transgenic plant, and 1~9 means the corresponding line; the subsequent numbering principle is the same).

Example 4. Identifying the virus resistance of T1 generation plants; using resistant plants (T0 and T1 generation) as rootstocks, grafting wild-type plants; inoculating CMV virus, and detecting the grafted plants that obtained virus resistance.

Step 1. Challenge screening of T1 generation

Because in the present invention, tomato seeds are reserved in a self-propagation mode, there will be self-recombination and separation phenomena in the T1 generation tomato. Therefore, molecular identification and virus inoculation identification of T1 generation seeds are necessary. We used the T0 generation of resistant plants as test materials, obtained seeds, sowed and propagated to obtain T1 generation plants, and identified transgene-positive plants among them by a rapid PCR method for subsequent virus resistance identification, according to the method in step 2 of Example 3 Inoculate CMV and identify its antiviral ability. The results are as follows:

resistance MM T11A.1 T12A.1 T12B.1 T13A.1 T1CP.1 T1CMV5.1 High resistance (HR) - 13 11 13 7 8 13 disease resistance (R) - - 2 2 1 - 1 Moderate resistance (MR) - 1 1 - - - 2 Sickness (S) - - 1 - - - - High Sensitivity (HS) - - - - - - - total 20 14 15 15 8 8 16

In the present invention, through the CMV challenge of T1 generation plants, it is found that although the resistance traits are separated, most positive T1 plants also have high CMV resistance.

Step 2. Transgenic T0 and T1 generation plants are used as rootstocks to study the resistance of grafted plants to CMV

In order to verify whether CMV resistance can be maintained in the scion, the present invention uses high-resistant transgenic T0 and T1 as rootstocks to graft susceptible varieties (MoneyMaker; MM). Grafting method, as shown in Figure 11.

For the T0 generation, we selected cutting seedlings of all high-resistant plants, and propagated 5 plants for each line, and used them as rootstocks. Graft non-transgenic plants, susceptible varieties (MM) scions. When the buds grow to 3-4 leaves open, begin to carry out virus inoculation identification according to the inoculation system method of cutting seedling CMV virus. For the T1 generation, because there are too many strains in the progeny, we only selected some strains for the experiment.

The grafting operation is as follows:

Reference (Shaharuddin, N.A., Han, Y., Li, H. and Grierson, D. 2006. The mechanism of graft transmission of sense and antisense genes silencing into matoplants. FEBS Lett. 28-29, 6579-86.)

(1) Cultivate tomato plants. After the rootstock grows to a height of 20-30 cm, cut off the top bud (about 5 cm), and use a blade to split the opening about 1.5-2 cm long from the top of the cut.

(2) Take the buds of the scion variety (about 3-5cm), and use a blade to cut the stem downwards into a wedge shape at the place where 2 true leaves are left at the top. Immediately insert the scion into the rootstock incision, insert it tightly with slight force, and fix it with grafting clips (or wrap it with plastic wrap).

(3) Cover the grafted plants with disposable plastic bags or put them in a plastic airtight shed to keep the humidity around the plants. After grafting, pay attention to observe the temperature, room temperature and humidity. The temperature is kept at about 23-25°C, and the humidity is kept at 85%-90%.

(4) After 3 to 4 days of grafting, start to gradually ventilate (open the plastic bag gradually). 5 to 10 days after grafting, the grafting wound is completely healed, and the heart leaves of the scion have the phenomenon of "spitting water". After 10 days, carry out normal management. Fertilizing water, strengthening the control of insect pests and leaf diseases, etc. During the growth of grafted plants, side buds will germinate in the leaf axils of the true leaves of the rootstock, and these side buds should be wiped off in time to prevent the side buds from having adverse effects on the scion.

The results showed that (as shown in the table below), different grafted lines of the T0 generation showed different virus resistance, and most of them were highly resistant. However, compared with the transgenic plants (rootstock), the CMV resistance of some corresponding grafted plants (scions) has decreased, which may be related to the RNA silencing signal transmission efficiency of different plants, the copy number of transgene inserts (or siRNA expression), graft healing. The degree is related to the growth state of the scion bud. The results of grafting showed that grafting susceptible scion varieties with highly resistant CMV virus transgenic plants as rootstocks can effectively improve the virus resistance of scion varieties. Highly resistant T1 rootstocks also imparted high virus resistance to scions (Fig. 10). It is further proved that the use of grafting technology by improving the rootstock is an effective method to improve the virus resistance of scion varieties.

Step 3. Molecular detection of rootstock and scion antibiotic resistance genes.

The method for improving plant virus resistance of the present invention is mainly realized by grafting transgenic rootstocks. At the same time, this method can cleverly avoid another major problem about the safety of transgenics, that is, to reduce or avoid the possibility of antibiotic resistance gene drift.

The present invention uses scion leaf as test material, takes transgenic plant leaf as contrast, extracts respective RNA, reverse transcription synthesizes cDNA, and uses it as template to detect the expression of NptII gene by the method of RT-PCR, verifies the expression of scion in the grafted strain. Genetically modified ingredients.

The result is shown in Figure 12, there is no transcript of NptII in the scion. Theoretically, without the gene transformation process in the scion, it is impossible to have the antibiotic resistance marker gene used in the plant expression vector. Therefore, it can be guaranteed that antibiotic resistance marker genes cannot exist in the sexual gametes produced by scion varieties.

110 Hunan Agricultural University

120 A method for obtaining virus resistance in scion varieties, RNA interference vector pCAMBIA2300-CP and transgenic method

130P11551NYDHN

16030

170PatentInversion3.3

2101

211276

212DNA

213 Fragment 1A

4001

tggatgcgtcctgtgcccgagggtattgtttatctgtcggttataatgaacgcggttta60

ggtccgaagtctgatggagagctttacattgtcaatagtgaatgcgtgatctgtaacagt120

gaatctttatccactgtcacgcgttctcttcaagctccaaccgggaccattagtcaagtt180

gacggagttgctggttgtgggaaaaccacggcaattaaatccatttttgagccgtccact240

gacatgatcgttaccgcgaacaagaagtccgcccaa276

2102

211277

212DNA

213 Fragment 2A1

4002

tgtccaacgccaaccatcgcgattcctccggatttaaaccgtgctactgatcgtgttgat60

atcaatttagttcaatccatttgtgactcgactctgcccactcgtagtattacgacgact120

cttttcatcaagtgttcgtcgaaagtgcagactattctatagatctggatcatgttagac180

ttcgacagtctgatcttattgcaaaaattccagattcagggcatatgataccggttctga240

acaccgggagcggtcacaagagagtaggtacaacgaa277

2103

211259

212DNA

213 Fragment 2B

4003

tggctcgtatggtggaggcgaagaagcagagacgaaggtctcacaaacagaatcgacggg60

aacgaggtcacaaaagtcccagcgagagagcgcgttcaaatctcagactattccgcttcc120

taccgttctatcaagtggatggttcggaactgacagggtcatgccgccatgtgaacgtgg180

cggagttaccccgagtctgaggcctctcgtttagagttatcggcggaagaccatgattttg240

acgatacagattggttcgc259

2104

211239

212DNA

213 Fragment 3A

4004

aaagacccttcagcatcagtggaaactgttcgagtaacagcacataacacttgagggaca60

ctcatgtatccttttgagcataattcaccaacatcatatccagacttaaagaaggaagca120

atacgaccgtgggttattcgggaacgaggggccggactgaaatagcattatcagcgcgc180

atccaatgatgccggcctaggtcacactcagtagccattttcttaatggcttcagggct239

2105

211211

212DNA

213 Fragment CP

4005

tttacggactgtcacccacacggtagaatcaaatttcggcaaaggattaactcgaatttg60

aatgcgcgaaacaagcttcttatcatattccgtgactgaatcaggtagtaacaacctttt120

accgtaataagaccccacggtctatttttggtggctttaggtaatagatgtgaacgtgta180

cccaggtctacagcgttcactccctacaaag211

2106

211600

212DNA

213 fusion fragment CMV

4006

ctgtgcccgagggtattgtttattctgtcggttataatgaacgcggtttaggtccgaagt60

ctgatggagagctttacattgtcaatagtgaatgcgtgatctgtaaatttaaaccgtgct120

actgatcgtgttgatatcaatttagttcaatccatttgtgactcgactctgcccactcgt180

agtattacgacgactcttttcatcaagcgtatggtggaggcgaagaagcagagacgaagg240

tctcacaaacagaatcgacgggaacgaggtcacaaaagtcccagcgagagagcgcgttca300

aatctcagactattccgcttcctaccgttctatcaagacccttcagcatcagtggaaact360

gttcgagtaacagcacataacacttgagggacactcatgtatccttttgagcataattca420

ccaacatcatatccagacttaaagaaggaagcaatacgaccgtgttcggcaaaggattaa480

ctcgaatttgaatgcgcgaaacaagcttcttatcatattccgtgactgaatcaggtagta540

acaaccttttaccgtaataagaccacggtctatttttggtggctttagggtaatagatg600

Claims (4)

1. a kind of method that makes scion kind obtain virus resistance, its step is as follows: (1) prepare antiviral transgenic stock; (2) grafting the scion to the transgenic rootstock to grow and develop; It is characterized in that: the anti-virus transgenic rootstock is obtained by transferring RNA interference vector, the target sequence of the RNA interference vector is a gene segment in the genome of the target virus, the target virus refers to cucumber mosaic virus CMV, and the Both the scion variety and the transgenic rootstock are tomato varieties, and the target sequence of the RNA interference vector is shown in SeqID No.5. 2. An RNA interference vector targeting the cucumber mosaic virus gene, characterized in that the target sequence of the RNA interference vector is shown in SeqID No.5. 3. transform the bacterial strain that has the RNA interference vector described in claim 2. 4. The strain according to claim 3, wherein the starting strain is transgenic Agrobacterium tumefaciens strain EHA105.