sRNA伴侣蛋白Hfq与sRNA RsmY对藤黄绿菌素合成途径转录激活子PltR表达的转录后调控机制

项目名称： sRNA伴侣蛋白Hfq与sRNA RsmY对藤黄绿菌素合成途径转录激活子PltR表达的转录后调控机制

项目编号： No.31470196

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 黄显清

作者单位： 上海交通大学

项目金额： 84万元

中文摘要： 藤黄绿菌素（Plt）是根际假单胞菌合成的一种广谱抗真菌抗生素。课题组前期研究表明，在假单胞菌株M18中存在独特而复杂的Plt合成调控网络。其中，在转录后水平，sRNA伴侣蛋白Hfq强烈负调控plt转录激活子PltR的表达，然而分子机理尚不清楚；sRNA RsmY转录后激活pltR表达，然而，RsmY对pltR的激活途径及机制，RsmY与Hfq在pltR表达调控中的相关性，均不清楚。本项目拟运用基因突变与过表达、lacZ报告分析、RT-PCR、Co-IP、RNA-seq、EMSA、足迹分析、SPR等技术，研究Hfq独自或协助其它反义sRNA对pltR负调控的分子机制；分析Hfq与RsmY的相互作用机制；比较研究RsmY结合的下游翻译阻遏蛋白RsmA与Hfq在pltR mRNA上的调控位点及机制。为阐明Plt合成的转录后调控通路奠定基础，探索sRNA的反义调控机制与结合调控蛋白机制之间的联系。

中文关键词： 假单胞菌；sRNA；伴侣蛋白Hfq；sRNA(小RNA)；抗生素合成；转录后调控

英文摘要： Pyoluteorin (Plt) is a broad-spectrum antifungal antibiotic produced by rhizosphere biocontrol Pseudomonas. Our extesive in-depth studies have shown that a unique and complex regulatory networks control Plt biosynthesis in Pseudomonas strain M18. The sRNA chaperone Hfq, at the post-transcriptional level, was shown to strongly down-regulate expression of the plt transcriptional activator PltR, however, the underlying molecular mechanism remains unknown. Also, the sRNA RsmY was found to post-transcriptionally activate the pltR expression; however, these remain unclear the activation pathway and mechanism by which RsmY controls the pltR expression and whether the sRNA RsmY is directly correlated with the sRNA chaperone Hfq in the regulation on pltR expression. In this study, through gene mutation or over-expression, lacZ report analysis, RT-PCR, Co-IP (Co-immunoprecipitation), northern blotting, RNA-seq (RNA sequecing), EMSA (Electrophoretic mobility shift assay), footprinting analysis, SPR (Surface plasmon resonance) technology, etc., we will study: (1) molecular mechanism by which Hfq, alone or in incollaboration with sRNA, reugulates on the pltR expression; (2) the interaction mechanism between the sRNA chaperone Hfq and the sRNA RsmY in the regulation of pltR expression; (3) comparison of target sites and regulatoy mechanism between the RsmY-binding downstream translational repressors RsmA and Hfq on the pltR mRNA. This study will lay the foundation for clarifying the post-transcriptional regulatory pathway and mechanism of Plt biosynthesis and exploring the connection between the sRNA-mediated antisense regulation and the sRNA-mediated gene regulation by sequestering regulatory proteins.

英文关键词： Pseudomonas;sRNA chaperone Hfq;sRNA(small RNA);Antibiotic biosynthesis;Post-transcriptional regulation