缺失GDD保守区的黄瓜花叶病毒复制酶基因的克隆及植物表达载体构建

Cloning of GDD deleted replicase gene of Cucumber mosaic virus and construction of its plant expression vector

以 CMV亚组 1株系 Fny- CMV RNA2基因组为模板 ,根据其序列设计引物进行 PCR扩增 ,得到 2 .5 kb的全长复制酶基因扩增产物 .对此产物进行纯化 ,并用 N co I和 Bsp HI进行双酶切 ,得到 3个片段 .将不含有 GDD保守区的 2个片段用 T4 DNA连接酶连接 ,并对连接产物进行 PCR扩增 ,得到 2 .2kb左右缺失 GDD保守区的黄瓜花叶病毒复制酶基因的扩增产物 .将其克隆到 p GEM- T Easy Vector上 ,进行序列测定 ,结果表明 GDD保守区确已缺失 .该缺失不导致开放阅读框架的移码 .将缺失 GDD保守区的基因定向克隆到植物表达载体 p BI12 1中 ,并经三亲交配导入根癌农杆菌中 ,经 PCR及酶切鉴定 。

The entire replicase gene of CMV subgroup 1 strain Fny was amplified by polymerase chain reaction (PCR). PCR products with length of 2.5 kb were digested with Nco I and Bsp HI, and three fragments were obtained. The two fragments which didn't encode GDD motif were ligated and amplified by PCR. The resulting 2.2 kb deleted replicase gene was cloned into pGEM T Easy Vector. The sequence analysis of the gene confirmed that GDD encoding region was deleted. The deletion of the GDD encoding region didn't cause frame shifting of the replicase open reading frame. The GDD deleted replicase gene was cloned into plant expression vector pBI121, and then transferred into Agrobacterium tumefaciens . PCR and digestion confirmed that the deleted replicase gene were transferred into A. tumefaciens .