苏云金芽胞杆菌RS24960基因功能研究

发布时间：2018-12-06 15:52

【摘要】：苏云金芽胞杆菌(简称Bt)是一种非常重要的生防微生物,具备靶标特异性强、对环境友好等特点,符合可持续发展的需求。但是Bt制剂也存在杀虫效果较慢、毒力有限以及田间应用不稳定的缺点,限制了Bt产品大规模推广。提高杀虫晶体蛋白含量或者提升芽胞形成率,从而达到提高产量的目的是提高其毒力行之有效的方法。本实验室前期在寻找母细胞裂解关键水解酶转录调节机制时发现一个具有高转录活性的启动子,转录组数据显示RS24960基因具有非常高的转录活性。利用β-半乳糖苷酶报告系统分析证实该启动子PRS24960是一个强启动子。在不同的sigma突变体中分析启动子的活性,发现该启动子受σE特异性控制。通过5'-RACE实验鉴定转录起始位点,表明转录起始是位于起始密码TTG上游55bp的碱基A。鉴于PRS24960具有高转录活性,利用启动子PRS24960指导cry1Ac基因表达,通过SDS-PAGE和Western blot实验检测所得Bt菌株Cry1Ac蛋白积累量及特异性,结果发现该启动子能与已报道的强cry类基因启动子Porf1cry8E以及cry1Ac基因本身启动子一样,高效地指导Cry1Ac蛋白的表达和积累,三个菌株间没有显著差别。进一步的杀虫活性检测表明,含不同启动子指导表达cry1Ac的菌株对二龄小菜蛾幼虫具有相似水平的杀虫活性。然而,透射电子显微镜(TEM)和原子力显微镜(AFM)观察菌株和晶体形态发现PRS24960与Porf1cry8E指导表达的Cry1Ac蛋白均不能组装成完美的双锥体晶体结构。此外,对RS24960基因功能进行研究。UniProt、STRING和NCBI数据库分析显示,RS24960编码一个含HTH-DEOR保守结构的小分子蛋白,但是其功能未知。利用同源重组方法在Bt HD73中敲除该基因获得突变株HD△RS24960,生长曲线测定发现突变株生长不受影响,而显微镜观察显示突变株芽胞形成和母细胞裂解时间提前,同时用FM4-64标记活细胞膜,在激光共聚焦显微镜下观察发现HD△RS24960在芽胞形成早期就与野生型有区别。此外,芽胞形成率测定发现突变株HD△RS24960的芽胞形成率升高。这些表型得到了遗传互补验证,从而证明RS24960在芽胞形成早期就已经参与芽胞形成调控。在大肠杆菌中成功表达RS24960蛋白,以期寻找该蛋白的作用靶标,进一步揭示其生物学功能。本文对RS24960基因功能及其启动子进行了分析,为利用非cry基因启动子成功指导cry基因表达提供了一个成功的例子,同时为鉴定RS24960基因功能,揭示苏云金芽胞杆菌芽胞形成调控机制提供了新的重要的依据,为今后芽胞杆菌遗传改良和应用提供了新的遗传资源和方法。
[Abstract]:Bacillus thuringiensis (Bt) is a very important biocontrol microorganism with high target specificity and environmental friendliness. However, Bt also has the disadvantages of slow insecticidal effect, limited virulence and unstable field application, which limits the large-scale popularization of Bt products. It is an effective method to improve the virulence of insecticidal crystal protein content or the formation rate of spores in order to increase the yield. A promoter with high transcriptional activity was found in our laboratory in search of the transcriptional regulation mechanism of the key hydrolase in mother cell lysis. Transcriptome data showed that the RS24960 gene had very high transcriptional activity. The 尾-galactosidase report system analysis confirmed that the promoter PRS24960 was a strong promoter. The promoter activity was analyzed in different sigma mutants and it was found that the promoter was controlled by 蟽 E specificity. The transcriptional initiation sites were identified by 5'-RACE experiments, indicating that transcriptional initiation is the base Abase of 55bp upstream of the initiation codon TTG. In view of the high transcriptional activity of PRS24960, the promoter PRS24960 was used to direct the expression of cry1Ac gene. The accumulation and specificity of Cry1Ac protein in Bt strain were detected by SDS-PAGE and Western blot experiments. The results showed that the promoter could effectively direct the expression and accumulation of Cry1Ac protein as well as the reported strong cry gene promoter Porf1cry8E and cry1Ac gene itself promoter. There was no significant difference among the three strains. Further detection of insecticidal activity showed that the strains expressing cry1Ac with different promoters had similar insecticidal activity against the second instar diamondback moth larvae. However, the transmission-electron microscopy (TEM) and atomic force microscopy (AFM) showed that the expression of Cry1Ac protein guided by PRS24960 and Porf1cry8E could not be assembled into a perfect double pyramidal crystal structure. In addition, the function of RS24960 gene was studied. UniProt,STRING and NCBI database analysis showed that RS24960 encodes a small protein containing conserved structure of HTH-DEOR, but its function is unknown. Homologous recombination method was used to detect the growth curve of mutant strain HD RS24960, by knockout of the gene in Bt HD73. The results showed that the growth of mutant strain was not affected, but the microscopical observation showed that the time of spores formation and mother cell cleavage of mutant strain was earlier. At the same time, the living cell membrane was labeled with FM4-64 and observed under confocal laser microscope. It was found that HD RS24960 was different from wild type in the early stage of spores formation. In addition, the spore formation rate of the mutant HD RS24960 was increased. These phenotypes were verified by genetic complementation, which showed that RS24960 had been involved in the regulation of spores formation in the early stage of spore formation. RS24960 protein was successfully expressed in Escherichia coli in order to find its target and further reveal its biological function. In this paper, the function of RS24960 gene and its promoter were analyzed, which provided a successful example for the successful use of non-cry gene promoter to guide the expression of cry gene, and to identify the function of RS24960 gene. Revealing the regulation mechanism of Bacillus thuringiensis spores provides a new important basis for the genetic improvement and application of Bacillus thuringiensis and provides a new genetic resource and method for the future application of Bacillus thuringiensis.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476

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