抗虫基因CryIA(b)植物表达载体的构建

Construction of Plant Expression Vector Containing Insect-resistant Gene CryIA(b)

苏云金芽孢杆菌杀虫晶体蛋白基因具有对鳞翅目昆虫的毒杀作用,因此被广泛用于转基因研究,以提高植物的抗虫能力。故以含有CryIA(b)基因的PKUB质粒为模板,利用PCR技术克隆出抗虫基因〔CryIA(b)〕,将其构建到PGEMT-Easy上,获得重组质粒PGEMT-CryIA(b),并测定全部序列,将PGEMT-CryIA(b)用BamH I和Sm aI酶切后插入表达载体PB I121的CaMV35S启动子和NOS终止子之间,构建成准备用于丽江云杉等针叶树种遗传转化CryIA(b)基因的植物表达载体PB I-CryIA(b),采用液氮冻融法将其转化到根癌农杆菌LBA4404和C58中,为下一步的转化工作奠定了基础。

Crystal protein from Bacillus thuringiensis has insecticidal function on Lepidoptera, its gene was therefore widely used in gene transfer to increase the pest resistance of plants. Taking the PKUB plasmid harboring insect resistance gene CryIA （b） as template, the CryIA （b） gene was obtained by PCR. Then the CryIA （b） gene was cloned into PGEMT-Easy vector, by which a new recombinant plasmid harboring the gene was got and the gene was sequenced. The fragment got from digesting the recombinant plasmid by BamHI and SamI was recovered and inserted into a site between CaMV35S promoter and NOS terminator of plant expression vector PBI121. By this way the plant expression vector PBI121-CryIA （b） was constructed. Then PBI121-CryIA （b） was introduced into Agrobacterium tumefaciens strain LBA4404 and C58 by freeze melting methods.