tmRNA靶向全局转录因子Crp介导维氏气单胞菌代谢重编程及氧化耐受

维氏气单胞菌是严重危害水产养殖的强毒病原菌，分布广、环境适应性强，其逆境耐受性是影响毒力的重要因素，但调控机制不清楚。tmRNA主要参与反式翻译核糖体拯救，对逆境耐受至关重要。基于前期研究我们提出细菌氧化耐受新型分子机制：tmRNA以不依赖反式翻译的sRNA形式负调控全局转录因子Crp，进而级联调控68个Crp下游靶标，介导代谢重编程，最终调节氧化耐受。本项目拟采用同源重组方法构建敲除株；用QPCR、双质粒报告基因系统和Western blot确定tmRNA对Crp，及Crp对下游靶标的调控方式；用凝胶迁移实验检测tmRNA与Crp mRNA，及Crp与靶标DNA的结合活性；根据所鉴定靶标的功能确定代谢重编程网络；进而解析tmRNA/Crp互作对氧化耐受的影响。本项目将拓展tmRNA作为sRNA功能研究的新领域，对阐明病原菌逆境耐受机制、寻找药物新靶点、防治细菌病害具有重要理论和实际意义。

Aeromonas veronii is a virulent pathogen that causes great threats to aquatic products, widely distributed and adaptable to various environments. Its resistance to stresses determines the virulence, although the mechanism remains unclear. Transfer-message RNA (tmRNA), which is known to be involved in the trans-translation ribosomal rescue system, plays an important role in stress tolerance in bacteria. Based on our previous studies, we propose a new action model regarding the oxidative resistance in the pathogen of A. veronii: tmRNA acts as an antisense sRNA to positively regulate the expression of global transcriptional regulator Crp, in dependent from its role in trans-translation, modulates the activities of 68 downstream targets in cascade reactions, mediates the reprogramming of core material and energy metabolism, and thus control the tolerance to oxidative stress. This project intends to use the homologous recombination strategy to construct deletion strains; use the techniques including real-time PCR, double-plasmid detection system, and Western blot to investigate the regulation modes between tmRNA and Crp, or between Crp and its downstream targets; use electrophoretic mobility shift assay (EMSA) in vitro to detect the binding activities between tmRNA and mRNA of Crp, or between Crp and the binding regions in the target DNAs; determine the reprogrammed metabolic network based on the functions of the identified targets; and thus to discover the final effects of tmRNA/Crp interaction on the tolerance to oxidative stress. The project will extend the study on the function of tmRNA as an antisense sRNA, and will provide theoretical and practical significance for uncovering the molecular mechanism of adversity adaptation in A. veronii, seeking novel therapeutic targets for bacterial disease treatments, and overcoming recurrent infections in aquaculture.