苏云金芽胞杆菌SigmaK因子的表达纯化及其调节子鉴定

发布时间：2018-06-14 09:29

本文选题：苏云金芽胞杆菌 + SigmaK因子　；参考：《中国农业科学院》2015年硕士论文

【摘要】：苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)属于芽胞杆菌属的蜡样芽胞杆菌群(B.cereus group),是一种特殊革兰氏阳性细菌,在产生芽胞的同时产生由杀虫蛋白组成的具有规则形状的伴胞晶体(主要由cry和cyt基因编码)。因其对靶标生物高效、对环境安全友好等特点而成为目前应用最广泛的微生物杀虫剂。在芽胞形成过程中,不同的Sigma(σ)因子在时空上级联起始基因转录,σK由sig K基因编码,是Bt芽胞晚期重要的Sigma因子,芽胞晚期主要涉及芽胞外壁形成、芽胞的成熟和释放、母细胞裂解、晶体的形成等重要细胞发育过程,目前对这些细胞过程所知仍然很少。因此本研究主要明确Bt中σK因子的调节子,为解析母细胞生长过程的晚期调控网络进而为阐明芽胞形成晚期细胞过程的分子机制奠定基础。以芽胞形成晚期全局调控因子ger E突变体作为对照,分别提取sig K和ger E突变体在T7、T10时期的总RNA进行DNA芯片分析,明确受σK控制的基因或操纵子。结果表明T7时期有158个受σK下调的基因和138个上调的基因。T10时期有878个受σK下调的基因和1317个上调的基因。利用σK因子结合位点的一致序列,分析T7和T10下调基因的启动子区域,发现264个基因的启动子具有σK因子结合位点,包括10个操纵子和242个基因。其功能包括氨基酸运输和代谢、碳水化合物运输和代谢、无机盐离子运输和代谢、信号转导、蛋白转译后修饰等其他功能和未知功能。表达并纯化了具有His标签的Sigma K蛋白,并对上述σK因子的调节子进行验证。前人研究已明确Sigma K控制cry1Ac基因的启动子转录,凝胶阻滞实验EMSA结果表明纯化的Sigma K蛋白可以与cry1Ac基因启动子结合,说明纯化的Sigma K具有体外与受其控制的启动子结合的功能。因此进一步通过EMSA实验验证了6个基因(芽胞萌发相关的基因HD73_3496,HD73_335水解酶基因HD73_3410和芽胞外壁相关的基因bxp B,exs B,bcl B)和一个操纵子(HD73_2493,HD73_2494)的启动子能与Sigma K因子结合,证明了这6个基因和1个操纵子受Sigma K因子控制。此外构建了水解酶基因HD73_3156的启动子和lac Z基因的融合载体,将载体转入HD73菌株及sig K突变体中,测定β-半乳糖苷酶活性发现HD73_3156的启动子活性在突变体中完全丧失,进一步证明了HD73_3156的转录受sK控制。
[Abstract]:Bacillus thuringiensis (BT) belongs to B.cereus group of Bacillus, which is a special Gram-positive bacteria. In addition to the production of spores, regular paracellular crystals composed of insecticidal proteins (mainly encoded by cry and cyt genes) were produced. It has become the most widely used microbial insecticide because of its high efficiency to target organisms and environmental safety. In the process of spore formation, different Sigma (蟽) factors are cascade initiation gene transcription in time and space. 蟽 K is encoded by sig K gene and is an important Sigma factor in the late stage of BT spores. The late stage of spores is mainly involved in the formation of the outer wall of the spores, the maturation and release of the spores. Some important cellular processes, such as mother cell cleavage and crystal formation, are still poorly understood. Therefore, in this study, the regulator of factor 蟽 K in BT was clarified, which laid a foundation for the elucidation of the molecular mechanism of late cell formation in the process of spores formation by analyzing the late regulatory network of the growth process of the mother cell. The total RNAs of ger K and ger E mutants at T7 / T10 stage were extracted from spores to identify the genes or operons controlled by 蟽 K, respectively. The results showed that there were 158 genes down-regulated by 蟽 K and 138 up-regulated genes at T7. At T10, there were 878 genes down-regulated by 蟽 K and 1317 genes up-regulated. By using the consistent sequence of 蟽 K factor binding sites, the promoter regions of T7 and T10 down-regulated genes were analyzed. It was found that 264 gene promoters had 蟽 K factor binding sites, including 10 operons and 242 genes. Its functions include amino acid transport and metabolism, carbohydrate transport and metabolism, inorganic salt ion transport and metabolism, signal transduction, protein post-translational modification and other functions and unknown functions. Sigma K protein with his label was expressed and purified, and the regulator of 蟽 K factor was verified. Previous studies have shown that Sigma K controls the promoter transcription of cry1Ac gene, and the results of gel block assay show that the purified Sigma K protein can bind to the promoter of cry1Ac gene, indicating that the purified Sigma K protein has the function of binding to the promoter controlled by Sigma K in vitro. Therefore, the promoters of six genes (HD733496 / HD73335 hydrolase gene HD733410 and bxp Bnexs Bbcl BCL BCL) and one operon HD732493 HD73SII 2494 were further verified by EMSA experiments to bind to Sigma K factor. It was proved that the six genes and one operon were controlled by Sigma K factor. In addition, a fusion vector of the promoter of the hydrolase gene HD73S3156 and the lac Z gene was constructed. The vector was transferred into the strain HD73 and sig K mutants. The activity of 尾 -galactosidase was determined and the promoter activity of HD733156 was completely lost in the mutant. It is further proved that the transcription of HD733156 is controlled by SK.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.1

【相似文献】