Construction of binary RNAi expression vector with virus resistance and genetic transformation in tobacco

【Objective】In order to develop new disease-resistant plant materials against two kinds of grape virus,【Method】The binary virus resistant RNAi expression vector was constructed using gateway technology.The conserved sequence of CP(coat protein) gene of Grapevine leafroll virus(GLRaV-3) and Grapevine fanleaf virus(GFLV) were obtained separately via RT-PCR.Then the two conserved sequences were connected to form the interference fragment of GLRaV-GFLV(508 bp) via overlapping PCR.And the fragment was cloned into the vector of pENTR/SD/D to form pENTR/SD/D-GLRaV-GFLV via TOPO cloning.Secondly the ccdB fragment on destination vector pH7GWIWG2(Ⅰ) was replaced by the interference fragment on pENTR/SD/D-GLRaV-GFLV via LR reaction to form the RNAi expression vector of pH7GWIWG2(Ⅰ)-GLRaV-GFLV which contained the two kinds of grape virus gene fragments.Then the vector was transformed into Agrobacterium strain EHA105 by alternate freezing and thawing method.And the RNAi vector was used to transform Nicotiana benthamiana.【Result】The objective fragments were transformed into tobacco successfully by PCR identification.【Conclusion】The Gateway technology-compatible construction of RNAi plant expression vector and tobacco transformation in this study make it possible to induce posttranscriptional gene silencing in the transformed plants and obtain novel double-virus-resistant plant materials.