TYLCCNV Cp Gene DsRNA Induces Tobacco Resistance To This Virus

N. benthamiana and TYLCCNV are the research materials in this dissertation. According to the reported TYLCCNV gene sequence, design specific primers and in the forward primers 5'to introduce restriction enzyme site BamHI, in the 3'to introduce restriction enzyme site XbaI; in the inverted primers 5'to introduce restriction enzyme site KpnI, in the 3'to introduce restriction enzyme site XhoI; obtain two including TYLCCNV-CP (458bp) gene fragments by PCR; reconstruct the pBin19 vector, which contains promoters and introns. Design specific primers, in the 5'end of the promoters, introduce restriction enzyme site XhoI; obtain Ro1C fragment by PCR. Obtain re-pBinl9 expression vectors by conventional molecular cloning technique, which contains four restriction enzyme sites BamHI,XbaI,XhoI and KpnI; pGM-T vector as a cloning vector. With conventional molecular cloning technique, the re-pBin19 expression vectors as a middle cloning vector, using introns Ro1A as a space, construct eukaryotic expression vectors containing TYLCCNV-CP (458bp) inverted-repeat gene fragments named CPF458+re-pBin19+CPR458. Furthermore, the recombinant plasmid was confirmed by restriction enzyme digestion,PCR,and sequencing. Through electroporation method, CPF458+re-pBin19+CPR458 is transformed into Agrobacterium LBA4404. And then in sterile conditions transformed the dsRNA interference vector CPF458+re-pBin19+CPR458 into tobacco(N. benthamiana) by Agrobacterium leaf-disc transformation, obtain a number of resistant plants by screening of resistance to kanamycin, and confirmed by PCR.This dissertation constructes an inverted-repeat eukaryotic expression vector and transformes into the N. benthamiana, induces tobacco resistance to TYLCCNV virus disease by dsRNA. In this way, it provides an effective method in preventing tobacco virus, and it has a significant meaning in ensuring safe production of tobacco.