Study on tobacco virus resistance with RNAi method
应用RNAi技术培育抗2种病毒病的转基因烟草

Total RNA of tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) were isolated and amplified using RT-PCR and ORF PCR methods.The conserved sequence of two virus among different strains were obtained using sequence alignment in NCBI GenBank; The fusion sequence for RNAi target sequence was built by inserting the interference sequence from CMV and TMV into the neighboring restriction site of pMD18-T.The PCR products with the forward and reverse primers was later subcloned into pUCCRNAi,and transferred into pCAMBIA2300-35S-OCS expression vector.Then introduced into tobacco varieties K326 by Agrobacterium mediated transformation.Three transformants which containing interference sequence of coat protein gene of TMV and CMV were obtained and approved by molecular identification.The results confirmed that RNA interference sequence from two virus have been introduced into Nicotiana tabacum successfully and the inhibition of RNA from virus in the infected tobacco were observed using real-time quantitative PCR.The data of disease screening found that the transformed tobacco showed resistance to TMV and CMV significantly.