苏云金芽孢杆菌cry2 Aa操纵元蛋白对杀虫蛋白晶体形成作用的研究

Function of Proteins Coded by Bacillus thuringiensis cry2 Aa10 Operon on ICPs Crystallization

利用重叠PCR的方法 ,通过两次PCR扩增 ,分别获得cry2Aa1 0操纵元的orf1、orf1 +orf2与cry2Ab5基因的融合片段。融合片段经BamHⅠ和EcoRⅠ双酶切与pHT31 5连接 ,分别构建了基因融合片段的原核表达载体pFU(orf1 +2Ab)和pFU(orf1 +orf2 +2Ab) ,电转化Bt无晶体突变株 4Q7后 ,扫描电镜下可观察到典型的方形晶体 ,通过SDS PAGE可检测到 6 0kD大小的蛋白表达带。结果表明 ,cry2Ab5可在cry2Aa1 0的启动子帮助下有效转录和表达 。

By using overlapping PCR techniques, cry2Ab5 was fusioned with orf1, orf2 and orf1+orf2 of cry2Aa10 , and then was inserted E. coili B. thutringiensis shuttle plasmid pHT315 respectively after digestion by Bam H1 and Eco R1. The recombinant plasmid was named as pFU (orf1+2Ab ), pFU (orf1+orf2+2Ab) and pFU( orf2+2Ab ) and electroporated into Bt acrystalliferous mutant strain 4Q7 respectively. Though the 60 kD protein band was detected in pFU( orf1+orf2+2Ab) and pFU (orf1+2Ab) by SDS PAGE, the square crystals was only observed in pFU (orf1+orf2+2Ab) by scanning electron microscope. The results showed that the orf1+orf2 from cry2Aa10 operon not only can help cry2Ab5 expression, but also assist the formation of square crystals.