小西葫芦黄花叶病毒外壳蛋白基因植物表达载体的构建

Construction of Plant Expression Vector of the Coat Protein Gene of Zucchini yellow mosaic virus from China

小西葫芦黄花叶病毒(Zucchiniyellowmosaicvirus,ZYMV)是危害中国葫芦科作物主要病毒之一。试验以该病毒中国分离物外壳蛋白基因的克隆载体pZCP-87(含ZYMV的CP基因)为材料,经SalⅠ和BamHⅠ双酶切,从胶上回收目的基因,与经过同两种酶酶切的植物表达载体pBIN438连接,转化感受态的大肠杆菌细胞,提取质粒,经PCR和SalⅠ/BamHⅠ双酶切验证,已将该病毒外壳蛋白基因克隆到植物表达载体上(重组质粒命名为pBZCP5),采用冻融法完成了对pBZCP5质粒的农杆菌转化。该工作是通过转基因获得抗ZYMV病毒研究的基础。

Zucchini yellow mosaic virus(ZYMV)is one of the prevalent viruses infecting cucurbits in China. The experiment was conducted with vector pPZCP-87 containing the coat protein gene of Zucchini yellow virus isolated in China. The vector was digested by SalⅠand BamHⅠand then ligased to plant expression vector pBIN438 digested by the same enzymes. The recombinant plasmid was transferred into competent cells of Escherishia coli DH 5α. The PCR and double digestion test showed that the coat protein gene was constructed into plant expression vector (recombinant plasmid designated as pBZCP5). The recombinant plasmid was transferred into competent cells of Agrobacterium tumefaciens LBA4404. One A. tumefaciens clone harboring recombinant plasmid pBZCP5 was obtained. Our experiments reported here is basic step in transgenic research for resistance to ZYMV.