LuxS/AI-2群体感应系统对水生拉恩氏菌HX2促生功能及根际细菌群落的调控研究

Quorum sensing is a cell-cell communication process in which bacteria response correlated to environment and act in unison by regulating gene expression synchronized in the group. LuxS/AI-2 quorum sensing system is useded for bacteria innerspecies and interspecies communication, has significant meaning in terms of regulating bacterial physiological function, signal conduction and enironmental adaptation and plays a crucial role in interaction of bacteria and plant. Rahnella aquatilis HX2 is a rhizobacteria that could well survive in the roots of maize and effectively promote maize growth, and these are related to the concentration of the inoculum. Genome sequence analysis showed that HX2 has the LuxS/AI-2 system and previous research in strain HX2 indicated that LuxS/AI-2 system is related to plant growth promoting. In this study, we will disrupt luxS、lsrK and tqsA gene which is response to AI-2 signal synthesis and transportation, and then analyze the effect of LuxS/AI-2 system to HX2 enironmental adaptation and plant growth promoting, such as PQQ, GDH or IAA producing. Simultaneously proteomic analysis will be performed to investigate the regulatory pathways of LuxS/AI-2 system by result of compared proteome-changes between HX2 and LuxS/AI-2 system mutant strains. Also, we will focus on the influence of LuxS/AI-2 quorum sensing circuit to the rhizosphere bacterial community by molecular ecology methods. The main aim of this study is to explore new methods to manager the rhizosphere bacterial community and provide scientific basis for plant growth promoting rhizobacteria (PGPR) application in agricultural production.

LuxS/AI-2群体感应系统是细菌种内及种间细胞交流的重要机制，在调节细菌生理功能、信号交流及环境适应方面具有重要意义。水生拉恩氏菌HX2（Rahnella aquatilis）能良好地定殖于玉米根部，并有效促进玉米生长。本项目重点研究植物根际促生菌HX2中LuxS/AI-2群体感应系统调节自身的生理代谢、适应定殖环境并与根际微生物进行互作交流机理。通过定位突变HX2菌株LuxS/AI-2信号分子合成与转运关键基因luxS、lsrK和tqsA，利用转录报告载体研究LuxS/AI-2系统对环境因子的感应及对促生相关基因转录的分子调控机制，利用基因组信息和蛋白质组技术研究HX2菌株LuxS/AI-2系统调控途径，并采用分子生态学方法研究LuxS/AI-2系统对根际土壤细菌群落的影响，探索根际细菌群落调控的新方法，为植物根际促生菌在农业生产上的更好应用提供科学依据。

LuxS/AI-2群体感应系统是细菌种内及种间细胞交流的重要机制，在调节细菌生理功能、信号交流及环境适应方面具有重要意义。水生拉恩氏菌HX2具有非常广泛的抑菌谱，耐酸性强，能产生PQQ、IAA和有机酸，溶解难溶磷，促进植物生长。研究结果表明，缺失pqq基因 （MH15）或gdh基因（MH16）的突变菌株在分泌有机酸、溶解矿质磷能力及促生效果明显低于HX2及其互补菌株。HX2菌株溶磷机理主要是产生葡萄糖酸，吡咯喹啉醌（PQQ）作为葡萄糖脱氢酶（GDH）的辅酶，共同参与了催化葡萄糖形成葡萄糖酸，分泌到土壤中后促进土壤难溶磷的溶解、玉米对磷的吸收和生长。HX2菌株对环境逆境的适应能力结果表明，对酸的耐受达到pH4.0，对盐的耐受达到1M NaCl，对温度耐受到37℃，对草甘膦、百草枯、Cu等重金属都具有较高的耐受性。定位突变了LuxS/AI-2系统三个关键基因luxS、lsrK和tqsA并进行了功能互补，同时将LuxS/AI-2系统突变体与LuxS/AI-2系统上游调控基因pqq基因和gdh基因，以及LuxS/AI-2系统下游调控基因phoQ基因，系统的研究了LuxS/AI-2系统在HX2菌株中对细菌促生功能和对环境条件感应的影响。结果表明，突变辅酶PQQ和葡萄糖脱氢酶（GDH）调控HX2菌株的LuxS/AI-2群体感应系统，突变pqq基因或者gdh基因后，HX2的AI-2群体感应信号合成显著降低。LuxS/AI-2群体感应系统在HX2中对其促生机理具有调节作用，其可以调控IAA、生物膜的产生以及细菌的运动能力，但不影响细菌的产细菌素、溶磷等性状。同时，通过实验还发现突变HX2的LuxS/AI-2群体感应系统后双组分调控系统phoP/phoQ中的phoQ基因转录发生影响，phoP/phoQ系统的激活对于细菌耐受酸性pH是必须的，为了适应不同的生态位，HX2的LuxS/AI-2群体感应系统通过调控phoP/phoQ系统对细菌耐受外界酸环境发挥着重要作用。研究结果对植物促生菌在田间应用，特别是在应用中通过调控菌株的环境逆境适应，扩大应用范围具有重要意义。