葡萄抗病毒双价RNAi植物表达载体构建及其对烟草的遗传转化

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

【目的】为获得兼抗2种葡萄病毒的抗病植物新材料,【方法】利用Gateway技术成功构建了葡萄抗病毒双价RNAi表达载体。先采用RT-PCR分别克隆获得了葡萄卷叶病毒(GLRaV-3)和扇叶病毒(GFLV)外壳蛋白基因保守区段,用重叠延伸PCR方法串联获得了干扰片段GLRaV-GFLV(508 bp);通过TOPO克隆将该片段克隆入载体pENTR/SD/D构建了入门克隆载体pENTR/SD/D-GLRaV-GFLV;再经过LR反应使入门克隆载体pENTR/SD/D-GLRaV-GFLV上的干扰片段GLRaV-GFLV替换目标载体pH7GWIWG2(Ⅰ)上的ccdB基因,形成了含2种葡萄病毒基因片段的RNAi表达载体pH7GWIWG2(Ⅰ)-GLRaV-GFLV,用冻融法将其转入农杆菌菌株EHA105,以此作为工程菌进行转化烟草,【结果】经PCR鉴定证明目的片段已成功转入烟草中。【结论】研究基于Gateway技术的RNAi植物表达载体的构建和对烟草的遗传转化,为诱导转基因植物转录后基因沉默、获得抗2种病毒病的植物新材料提供了可能。

【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.