摘要
以pUC19为母体，克隆了Btken－Ag（B.thuringiensissubsp.kenyaeAg）的复制起始区（～1．6kb）和pUC4K的aphⅠ基因，构建成穿梭载体pHV－1。pHV－1在E.coli中经100个世代，质粒保持率在80％以上；5在Bti4Q8（B．thuringiensissubsp.israelensis4Q8）中经40个世代，质粒保持率在80％以上。将B．licheniformis热稳定α－淀粉酶基因启动子控制下的Bt9510（B．thuringiensis9510）的crylC结构基因插入pHV－1，构建成重组质粒pHV－crylC，电激转化导入Bit4Q8。光学显微镜下观察，Bit4Q8无晶体产生；而Bit4Q8（pHV－crylC）则出现典型的菱形晶体，其晶体略小于Bt9510。生物测定结果表明表达的Cry1C蛋白对甜菜夜蛾具有一定的毒杀活性。
We have constructed the E. coli-Bt shuttle vector pHV-1 by cloning the replicon (~ 1.6kb) of Bt ken-Ag and the aphI gene of pUC4K into pUC19. The rate of plasmid maintenance is more than 80% after 100 generations in E coli, whereas 80% after 40 generations in Bti 4Q8. We have also constructed pHV-crylC through cloning the α-amylase promoter from B. licheniformis and the crylC gene from Bt 9510 into pHV-1 and introduced it into Bti 4Q8 by means of electroporation. Under the microscope, we can see that there is no crystal in Bti 4Q8, however, there are many rhomboid crystals in Bti 4Q8 (pHV-crylC), which are smaller than those of Bt 9510. The bioassay result of Bti 4Q8 (pHV-crylC) demonstrates that the expressed crystal protein is insecticidally active against Spodoptera exigue.
基金
天津自然科学基金!973607511
国家"九五"攻关项目!96-C01-0201
