群体感应系统阻遏抗真菌剂藤黄绿菌素生物合成的分子机制

项目名称： 群体感应系统阻遏抗真菌剂藤黄绿菌素生物合成的分子机制

项目编号： No.31270083

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： 黄显清

作者单位： 上海交通大学

项目金额： 82万元

中文摘要： 藤黄绿菌素(Plt)是根际假单胞菌合成的一种具有广谱抗真菌活性的抗生素。课题组针对假单胞菌株M18，前期已深入开展其Plt合成及调控的相关研究。其中发现三大假单胞菌群体感应(QS)系统(LasI/LasR、RhlI/RhlR、Pqs/PqsR)均强烈抑制Plt合成及基因表达。这些QS系统主要激活相关靶基因的表达，其转录激活机制也研究得较为透彻。然而，QS直接参与转录阻遏的分子机理尚不清楚。本项目拟运用DNA突变、lacZ报告分析、EMSA、SPR、RT-PCR等技术，比较研究三大QS系统阻遏Plt合成基因表达的分子机理,包括：鉴定靶启动子及结合元件；分析应答调控子与靶DNA的相互作用机制；评估信号分子在QS阻遏调控中的必要性与分子功能；剖析抑制Plt合成的QS级联调控通路；探索受PqsR阻遏的Px启动子的功能。为阐明Plt合成调控机制与网络奠定基础，为诠释细菌QS阻遏调控机理作出贡献。

中文关键词： 假单胞菌；群体感应；藤黄绿菌素；次级代谢；转录调控

英文摘要： Pyoluteorin (Plt) is a broad-spectrum antifungal antibiotic produced by rhizosphere Pseudomonas. We have carried out extensive in-depth studies about Plt biosynthesis and its regulation in the Plt-producing Pseudomonas strain M18. Three Pseudomonas quorum sensing (QS) systems (Las,Rhl,PQS), which are respectively composed of signalling molecule synthase(s) (LasI, RhlI and PqsABCDE) and response regulator (LasR, RhlR and PqsR), were shown to strongly inhibit Plt biosynthesis and its gene expression in the strain M18. These three QS systems mainly activate the expression of relevant target genes, and their activation mechanism have also been clarified. However, the molecular mechanisms by which these QS systems directly exert their transcriptional repression on target genes remain unknown. In this study, we will comparatively study and thus clarify the molecular mechanisms involved in transcriptional repression of three QS systems on Plt biosynthesis and its gene expression through DNA mutation, lacZ report analysis, EMSA (Electrophoretic Mobility Shift Assay), SPR (Surface Plasma Resonance), RT-PCR, et al., including: (1) identifying and characterizing target promoters and other DNA elements; (2) analyzing the interaction mechanisms between response regulators and their target DNAs; (3) evaluating the necessity a

英文关键词： Pseudomonas；Quorum sensing；Pyoluteorin；Secondary metabolism；Transcriptional regulation