携带黄瓜花叶病毒外壳蛋白基因的双元载体构建

Construction of Binary Vector of Cucumber Mosaic Virus Coat Proteingene

以黄瓜花叶病毒外壳蛋白基因为目的基因,以大肠杆菌DH5α为表达载体体外操作寄主菌,农杆菌LBA4404为最终双元载体寄主菌。将RT-PCR获得的目的基因片段连接到pMD18-Tsi mple Vectoer上,冻融法转化到大肠杆菌扩繁后,经限制性核酸内切酶酶切插入表达载体适宜酶切位点上,重组质粒DNA经冻融法转化到只含辅助质粒的根癌农杆菌中得到工程菌,完成含目的基因的双元载体构建,为培育抗CMV病毒的番茄品种打下基础。

The coat protein gene of cucumber mosaic virus was as target gene;and the E. coli DH5a was as the host bacte- ria of the intermediate vector for vitro operation;and the Agrobacterium tumefaciens LBA4404 was as the host bacteria of the final binary vector in this paper. The target gene was connected to the pMD18-T simple Vectoer after RT-PCR, than the recombinant plasmid was transformed into E. coli by freeze-thaw method, which was insert into expression vector restriction sites after digested with restriction endonuclease. The new recombinant plasmid DNA was transformed into the Agrobacterium bacteria which contain only helper plasmid by the freeze-thaw method. Than the construction of binary vector of target gene was completed.