Study On Improving The Antiviral Complex Infection Ability Of Tomato By Double Targeting RNAi

Tomato is tomato genus of the Solanaceae plant.As edible fruits and vegetables in the world,tomatoes are widely grown.Tomato virus disease has affected their yield and product quality seriously.Tobacco mosaic virus and cucumber mosaic virus occupy an important position among the 10 kinds of main tomato virus source, which widespread harm the world tomato planting areas,by the form of single infection or compound infection.The current common prevent method is cultivating disease resistant varieties or breeding virus-free seedlings. As one of the genetic engineering technologies,RNA interference technology has shown its advantages gradually, which is becoming one of the most popular means of breeding for disease resistance concern,because of its high specificity, hereditability and biological safety.On the basis of RNA interference mechanism to construct dual targeting RNA interference vector systemfor TMV and CMV.The method is as follows: with TMV and CMV virus gene as RNA interference target genes,to design the target gene target sequence primer according to the gene homology between the virus strains.Extract total RNA from tomato leaves,which have infected by TMV and CMV.Obtain the purpose sequence cDNA of TMV and CMV by RT-PCR.With the help of T vector,connect the two target sequencesed into a DNA sequence.Connect it to the cloning site of PUCC-RNAi intermediate vector by forward and backward insertion after enzyme digestion,becoming the bidirectional repetitive structure,with the form of the target sequence in vectorin; After enzyme digestion, insert into pC2300-35S-OCS expression vector.Recombinant RNAi expressing vector plasmid were transformed into Agrobacterium by electroporation, completing the construction of RNAi binary vector.Selecting the leaves and hypocotyls of tomato aseptic seedling as explants,by Agrobacterium mediated method to infect tomato explants,then the explants were cultured. Screen 25 resistant plants in antibiotic selection culture medium, PCR methods were detected by specific primers PCR and 35 S promoter,7 transgenic tomato seedlings had been obtained.Tomatoes are artificially inoculate with tobaccomosaic virus and cucumber mosaic virusand investigate the disease condition. The result shows that the disease resistant ability of transgenic tomato were significantly higher than the control group.Choosing 3 transgenic tomato seedlings with disease resistance as experimental group and 2 common tomato seedlings as the control group.Extract total RNA from plants,which was detected by one-step fluorescence quantitative PCR.The results are as follow:after virus inoculation of transgenic tomatoes,the mRNA expression quantity of TMV and CMV genes in transgenic tomatoes are significantly lower than the control varieties,the result of investigation shows that,the resistance of RNAi transgenic tomato to TMV and CMV is based on degradation of the transcription product of virus gene by RNA interference.