苏云金芽胞杆菌cry8Ea启动子区orf1片段缺失增强启动子活性

Deletion of the orf1 fragment from the cry8Ea promoter region enhances the transcriptional activity in Bacillus thuringiensis

本文分别构建了cry8E基因上游的启动子(Porf18E)和其上游缺失orf1基因的启动子(PΔorf18E)融合lacZ基因的表达载体,通过β-半乳糖苷酶活性的分析,发现PΔorf18E的转录活性高于Porf18E。分别用PΔorf18E和Porf18E指导cry1Ac基因的表达,通过光学显微镜观察,发现两个启动子指导表达的Cry1Ac蛋白均可形成双锥形晶体;通过总蛋白定量分析发现,缺失orf1基因的启动子(PΔorf18E)指导的Cry1Ac蛋白表达量高于Porf18E启动子指导的Cry1Ac蛋白表达量;生物活性测定表明:PΔorf18E指导的Cry1Ac晶体蛋白对小菜蛾Plutella xylostella具有杀虫活性,高于Porf18E指导的Cry1Ac晶体蛋白对小菜蛾的杀虫活性。本文获得的强活性的启动子PΔorf18E是目前已报道的转录活性最高的cry基因启动子,该启动子为Cry蛋白的表达和遗传工程菌株构建提供了重要元件。

The cry8E promoter (Porf18E) and the promoter (PΔorf18E) with the deletion of orf1 fragment, located upstream of cry8E , were fused with lacZ gene. The transcriptional activity of PΔorf18E was higher than that of Porf18E. The expression of Cry1Ac was directed by PΔorf18E and Porf18E, and the strains HD -(Porf18E-1Ac) and HD -(PΔorf18E-1Ac) were obtained. Both strains could produce the typical bipyramidal crystals, while HD -(PΔorf18E-1Ac) strain produced more Cry1Ac, which was more toxic to Plutella xylostella than HD -(Porf18E-1Ac). PΔorf18E has the highest transcriptional activity in cry gene promoters. It provides an important element for construction of engineered Bt strains.