嗜热链球菌全局转录调控因子CodY对Ⅱ型CRISPR/Cas系统的调控机制研究

项目名称： 嗜热链球菌全局转录调控因子CodY对Ⅱ型CRISPR/Cas系统的调控机制研究

项目编号： No.31501454

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 农业科学

项目作者： 陆文伟

作者单位： 江南大学

项目金额： 21万元

中文摘要： 嗜热链球菌是乳制品发酵的重要生产菌株，进化过程中，菌株获得多种抗性机制抵制噬菌体等外源DNA的侵染。CRISPR/Cas系统是广泛存在于原核生物中的新型抗性机制，赋予嗜热链球菌抵抗外源DNA入侵的能力，同时使菌株可以通过基因横向转移获得外源基因来适应牛奶环境。预示在嗜热链球菌体内存在着精细调控CRISPR/Cas表达的机制，维持菌体在取舍外源DNA 的平衡。前期转绿组分析发现，全局转录调控因子CodY的缺失引起Ⅱ型CRISPR/Cas的转录提高。基于此，本项目拟通过比较基因组分析研究嗜热链球菌ST2017 CRISPR/Cas系统的遗传组成；结合基因缺失突变和荧光定量PCR确定其表达模式；通过DNA-蛋白相互作用技术，阐明CodY调控作用的分子机制；进一步通过转录组学和蛋白质差异表达分析，探索CodY调控靶点Cas9蛋白的潜在功能。为嗜热链球菌理性选择外源DNA的分子进化机制提供新认识。

中文关键词： 嗜热链球菌；CRISPR/Cas；系统；全局转录调控因子；代谢调控；乳酸菌

英文摘要： Streptococcus thermophilus is a major dairy starter traditionally used in the manufacture of yogurt and cheeses. In the long process of evolution, the strain evovled diverse defense mechanisms that allow them to withstand viral predation and exposure to invading nucleic acid. In many Bacteria, including S. thermophilus, clustered regularly interspaced short palindromic repeats (CRISPR) and cas gene cluster, which provide acquired immunity against viruses and plasmids by targeting nucleic acid in a sequence-specific manner. Meanwhile, the strain could obtain functional genes through lateral gene transfer to adapt to the milk environment. Preliminary RNA-seq result showed that the lack of global regulator CodY increased the transcription level of CRISPR/Cas type-II system. This project intends to determine the genetic composition of type-II CRISPR/Cas system in S. thermophilus ST2017 by comparative genomic analysis, reveal the expression pattern of CRISPR/Cas type-II system in combination with gene deletion and real-time quantitative PCR, elucidate the regulation mechnism of CodY, and explore the potential function of Cas9 protein through transcriptome and proteomic analysis. The results of this study may provide a new perspective to understand the role of CodY in the rational choice of exogenous DNA as the molecular evolution mechanism of S. thermophilus.

英文关键词： Streptococcus thermophilus;CRISPR/Cas system;global regulator;Metabolic regulation;lactic acid bacteria