pBIN438-CMV△Rep表达载体的构建及其在根癌农杆菌中的转化

摘要

用PCR方法扩增黄瓜花叶病毒部分复制酶基因（CMV△Rep），连接到PUCm-T载体上构建成克隆载体PUCm-T-CMV△Rep。用BamHⅠ和SalⅠ分别对克隆载体PUCm-T-CMV△Rep和植物表达载体pBIN438进行双酶切，获得目的片段和线性质粒。在T4 DNA连接酶的作用下进行定向连接，构建成植物表达载体pBIN438-CMV△Rep。采用CaCl2冻融法将重组子导入根癌农杆菌LBA4404。经PCR和双酶切鉴定，表明重组质粒pBIN438-CMV△Rep已成功导入根癌农杆菌中。%Cucumber mosaic virus partial replicase gene were amplified by PCR(CMV Rep),connected to the PUCm-T vector to construct the cloning vector of PUCm-T-CMV Rep. With BamH I and Sal I of PUCm-T-CMV cloning vector Rep and the plant expression vector pBIN438 were digested,obtained fragment and linear plas⁃mid. Directional connection in T4 DNA ligase,a plant expression vector was constructed by pBIN438-CMV Rep. Using CaCl2 freeze-thaw method the recombinant plasmid into Agrobacterium LBA4404. By PCR and double enzyme diges⁃tion showed that the recombinant plasmid,pBIN438-CMV Rep has been successfully introduced into Agrobacteri⁃um tumefaciens.