Cloning of GDD deleted replicase gene of Cucumber mosaic virus and construction of its plant expression vector
Wen, R.I.; Zheng Zhong; Zhou XuePing
Journal of Zhejiang University Agriculture and Life Sciences 27(5): 531-534
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 NcoI and BspHI, and three fragments were obtained. The two fragments which did not encode the 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 the 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.