苏云金芽胞杆菌高效表达载体的构建

Construction of high-level expression vector for Bacillus thuringiensis

摘要:

【目的】通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性, 筛选出一个强启动子, 利用强启动子构建一个苏云金芽胞杆菌(Bacillus thuringiensis, 简称Bt)高效表达载体。【方法】利用启动子融合lacZ技术检测了4种启动子的转录活性。通过扫描电子显微镜观察晶体、SDS-PAGE、蛋白定量和生物活性测定等方法对新建高效表达载体进行功能验证。【结果】构建了Pcry1A、Pcry3A、Pcry4A和Pcry8E 4个启动子融合报告基因lacZ的表达载体, 经β-半乳糖苷酶活性分析得知, 启动子活性从高到低依次为Pcry8E>Pcry1A>Pcry4A>Pcry3A。选取cry8E启动子, 以pHT315作为基础载体构建苏云金芽胞杆菌高效表达载体pHT315-8E21b, 将cry1Ac基因连接到pHT315-8E21b和广泛应用的cry3A启动子指导的pSXY-422b上, 分别转入无晶体突变株HD-73?, 获得菌株HD-8E1Ac和HD-422-1Ac。扫描电子显微镜观察显示, HD-8E1Ac菌株可以形成菱形晶体, 说明正确表达了cry1Ac基因。SDS-PAGE分析结合蛋白定量实验表明pHT315-8E21b表达效率高于pSXY-422b。对小菜蛾(Plutella xylostella)的生物活性测定表明HD-8E1Ac菌株对小菜蛾有生物活性, 且菌株活性高于HD-422-1Ac。【结论】利用强启动子Pcry8E构建了一个能在Bt中高效表达的穿梭载体pHT315-8E21b, 该载体可正确表达cry1Ac基因, 其表达效率高于被广泛应用的pSXY422b。

Abstract:

[Objective] To construct a vector for high-level expression of cry genes in Bacillus thuringiensis, a strong promoter was screened by comparing the transcriptional activities among cry1A, cry3A, cry4A and cry8E promoters. [Methods] The transcriptional activities of the four promoters were detected by fusing with lacZ gene. Scanning electron microscopy was used to detect the abilities of crystal protein formation. SDS-PAGE, protein quantitation and bioassay were performed for the functional verification of the high-level expression vector. [Results] The four promoters Pcry1A, Pcry3A, Pcry4A and Pcry8E were fused with lacZ report gene. Beta-galactosidase assay showed that the activities of the four promoters were, in decreasing order, Pcry8E>Pcry1A>Pcry4A>Pcry3A. The cry8E promoter was selected to construct the high-level expression vector pHT315-8E21b based on plasmid pHT315. cry1Ac gene was inserted into both pHT315-8E21b and the widely used plasmid pSXY-422b which was initiated by cry3A gene promoter, respectively. The two expression plasmids were introduced into the acrystalliferous mutants HD-73?, and HD-8E1Ac and HD-422-1Ac strains were obtained. HD-8E1Ac and HD-422-1Ac strains Cry1Ac protein was observed by scanning electron microscopy, the results indicated that HD-8E1Ac strain produce diamond crystals compared with HD-73?. SDS-PAGE and protein quantitation analysis showed that the expression of cry1Ac gene is significantly increased in HD-8E1Ac compared with that in HD-422-1Ac. Bioassay results showed that HD-8E1Ac possesses much higher toxic against Plutella xylostella compared with HD-422-1Ac. [Conclusion] The high-level expression vector pHT315-8E21b initiated by cry8E gene promoter has the ability to express Cry1Ac correctly. It has higher efficiency of expressing insecticidal crystal protein compared with the widely used plasmid pSXY-422b.