福氏志贺菌2a型301株uhpT突变体的构建及功能分析

发布时间：2018-05-25 17:29

本文选题：基因敲除 + uhpT　；参考：《沈阳药科大学》2009年硕士论文

【摘要】： 本文以基因敲除方法研究志贺氏菌的功能基因,以RecA重组系统为基础,采用低拷贝自杀质粒pCVD442作为本敲除体系的重组载体,为了克服该质粒在进行分子克隆操作中的困难,将Gateway技术应用在敲除前期的重组质粒构建过程中,成功构建了福氏志贺菌2a型301株uhpT基因部分缺失的突变体MT,从而建立了在福氏志贺菌中进行定位插入或缺失突变的敲除技术平台。对突变株分别在培养基、细胞水平进行了功能检测。在普通LB培养基及TSB培养基中突变株与野生株的生长没有显著差异；突变株在U937细胞的胞内存活增殖能力与野生株没有显著差异,在HeLa细胞的胞内存活增殖能力突变株比野生株下降了12.7%。这说明uhpT基因在志贺氏菌的胞内存活增殖中仍然具有一定的作用。上述结果提示志贺氏菌中uhpT基因与细菌在细胞内的存活和增殖有关,可能由于细菌中其它碳源物质转移酶系统的存在代偿了敲除基因的功能缺陷,因此突变体在生长水平没有特别显著的变化。利用Gateway技术与自杀质粒相结合的敲除体系具有高效高通量的特点。本研究建立了一种对福氏志贺菌功能基因组研究有效的基因敲除技术平台,并且提供了对志贺氏菌中未知功能基因研究值得参考的新思路。
[Abstract]:In this paper, the functional gene of Shigella was studied by gene knockout method. Based on the RecA recombination system, the low copy suicide plasmid pCVD442 was used as the recombinant vector of the knockout system. The Gateway technique was applied to the construction of recombinant plasmid in the early stage of knockout. The mutant MTT with partial deletion of uhpT gene of Shigella flexneri strain 2a was successfully constructed, and a knockout technique platform for localizing insertion or deletion mutation in Shigella flexneri was established. The function of the mutant was detected in medium and cell level respectively. There was no significant difference between the growth of mutant and wild strain in common LB medium and TSB medium, but there was no significant difference in survival and proliferation ability between mutant and wild strain in U937 cells. The ability to survive and proliferate in HeLa cells decreased by 12. 7% as compared with wild strains. This suggests that uhpT gene still plays a role in the intracellular survival and proliferation of Shigella. These results suggest that the uhpT gene in Shigella is related to the survival and proliferation of bacteria in cells, which may be due to the functional deficiency of the knockout gene due to the existence of other carbon-source transferase systems in the bacteria. Therefore, there was no significant change in the growth level of the mutant. The knockout system using Gateway and suicide plasmids has the characteristics of high efficiency and high throughput. In this study, an effective gene knockout platform was established for the study of Shigella flexneri functional genomes, and a new idea for the study of unknown functional genes in Shigella flexneri was provided.
【学位授予单位】：沈阳药科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

【相似文献】