嗜盐古菌CRISPR/Cas系统与基因组稳定性机制

项目名称： 嗜盐古菌CRISPR/Cas系统与基因组稳定性机制

项目编号： No.31271334

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 向华

作者单位： 中国科学院微生物研究所

项目金额： 110万元

中文摘要： 以极端嗜盐古菌特有的Hmari亚型CRISPR/Cas系统为研究对象，通过基因敲除、CRISPR元件突变、Northern杂交、引物延伸等分子生物学技术解析CRISPR/Cas系统在嗜盐古菌中的转录调控机制、crRNA前体加工机制，以及相关Cas蛋白的生理生化功能；通过组合基因敲除/互补分析、Microarray分析、全基因组MFA分析，结合电镜观察、流式细胞仪、脉冲场电泳分析等，探索Cas6缺失引起嗜盐古菌生长和基因组稳定性变化的深层机制；构建嗜盐古菌新的病毒-宿主互作系统，分析嗜盐古菌CRISPR/Cas系统抗病毒机制与其基因组稳定性之间的关系。最终为深入理解Hmari亚型CRISPR/Cas系统的生理功能、作用机制及其与嗜盐古菌基因组复制与进化的关系提供新的知识和见解。

中文关键词： 嗜盐古菌；CRISPR-Cas；引发适应；病毒；基因组稳定性

英文摘要： This project will mainly focus on the mechanism of the Hmari-subtype CRISPR/Cas system functioned in virus immunity and genome stability in the extremely halophilic archaea. First, the transcription regulation of the CRISPR, processing of the crRNA precursors, physiological and biochemical functions of the Cas proteins, will be determined by means of gene knockout, CRISPR element mutation, Northern blotting, primer extension and other molecular biology techniques. Second, the mechanisms of the impact of Cas6 (and other associated proteins) on the genomic stability as well as the cell growth will be addressed by gene knockout and complementation, Microarray, genome-wide marker frequency analysis (MFA), electron microscopy, flow cytometry analysis etc. Third, a novel halophilic archaea-virus interaction system will be established, and will be used to investigate the function and relationship of the haloarchaeal CRISPR/Cas system involved in anti-virus and genome stability. Taken together, this project will provide novel knowledge of the physiological and biochemical functions of the Hmari-subtype CRISPR/Cas system, and insights in understanding the impact and mechanism of the CRISPR/Cas system on genome replication and stability in haloarchaea.

英文关键词： Haloarchaea；CRISPR-Cas；priming adaptation；virus；genome stability