抗虫Cry2Ab基因的原核表达及玉米的遗传转化

Prokaryotic Expression of Insect-resistant Cry2Ab Gene and Genetic Transformation of Maize

玉米的产量深受虫害的影响,抗虫Cry2Ab基因对玉米黏虫抗性有重要意义。利用外源抗虫基因Cry2Ab构建到原核表达载体pET-22b中,对其进行SDS-PAGE检测和抗黏虫性鉴定。SDS-PAGE检测结果表明,Cry2Ab基因编码的杀虫蛋白在大肠杆菌BL21中得到正确的表达,条带大小为70.68 kD;抗黏虫性鉴定结果表明,当Cry2Ab蛋白浓度为100μg/g时,对东方黏虫致死率达到86.66%。同时构建pCAMBIA3301-Cry2Ab-Bar植物表达载体,通过农杆菌介导法将外源抗虫基因Cry2Ab转入玉米自交系GSH9901愈伤组织中,利用除草剂筛选及PCR技术检测得到T;转基因阳性植株4株。获得的转基因抗虫玉米植株为抗虫转基因玉米的研发及抗虫玉米新种质的创制奠定了基础。

The yield of Chinese corn is deeply affected by insect pests.Therefore,dozens of transgenic corn lines derived from the soil bacterium Bacillus thuringiensis(Bt)insecticidal protein have been developed,and Cry2Ab is more resistant to oriental armyworm than other Cry2 proteins.Therefore,in this study,the exogenous insect-resistant Cry2Ab gene was used to construct the prokaryotic expression vector pET-22 b,and SDS-PAGE detection and identification of resistance to armyworm were carried out.SDS-PAGE analysis showed that the insecticidal protein encoded by the Cry2Ab gene was correctly expressed in E.coli BL21 with a band size of 70.68 kD.The identification of resistance to armyworm showed that when the concentration of Cry2 Ab protein was 100μg/g,the fatality rate reached 86.66%.At the same time,pCAMBIA3301-Cry2 Ab-Bar plant expression vector was constructed,and the exogenous insect-resistant gene Cry2Ab was transferred into maize inbred line GSH9901 callus by Agrobacterium-mediated method.T;transgenic positive plants were obtained by herbicide-screening and PCR detection strain.The genetically modified insect-resistant corn plants obtained in this study lay foundation for the development of insect-resistant transgenic corn and the creation of new insect-resistant corn germplasm.