论文部分内容阅读
【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。
【Objective】 A strong promoter was screened by comparing the promoter transcriptional activity of four genes cry1A, cry3A, cry4A and cry8E, and a strong promoter for Bacillus thuringiensis (Bt) expression vector was constructed. 【Method】 The transcriptional activities of four kinds of promoters were detected by promoter fusion lacZ technique. The new high efficient expression vector was verified by scanning electron microscopy (SEM), SDS-PAGE, protein quantification and bioassay. 【Result】 The expression vector lacZ containing 4 promoters of Pcry1A, Pcry3A, Pcry4A and Pcry8E was constructed. According to the analysis of β-galactosidase activity, the promoter activity from high to low was Pcry8E> Pcry1A> Pcry4A > Pcry3A. The cry8E promoter was selected to construct pHT315-8E21b plasmid with pHT315 as the basic vector. The cry1Ac gene was ligated into pHT315-8E21b and pSXY-422b, which is widely used by cry3A promoter. HD-73®, strains HD-8E1Ac and HD-422-1Ac were obtained. Scanning electron microscopy showed that the HD-8E1Ac strain could form rhombohedral crystals, indicating that the cry1Ac gene was correctly expressed. SDS-PAGE analysis of binding protein quantitative experiments showed that the expression efficiency of pHT315-8E21b was higher than that of pSXY-422b. The bioassay of Plutella xylostella showed that the strain HD-8E1Ac was bioactive to Plutella xylostella and its activity was higher than that of HD-422-1Ac. 【Conclusion】 The shuttle vector pHT315-8E21b, which can express efficiently in Bt, was constructed by strong promoter Pcry8E. This vector can correctly express cry1Ac gene and its expression efficiency is higher than that of pSXY422b, which is widely used.