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

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 .