促生菌SQR9黄瓜根际趋化信号筛选及其受体蛋白鉴定

Plant growth-promoting rhizobacterias (PGPRs) are a group of bacteria that can promote plant growth and control soil-borne diseases. Application of PGPRs as microbial fertilizers is a promising approach for reducing chemical fertilizer input and promoting the sustainable development of agriculture in China. Effective colonization of the applied PGPRs in rhizosphere is prerequisite for exerting the roles of plant growth promotion and suppression of soil-borne diseases, which is based on the chemotaxis reaction of PGPRs towards plant roots induced by root exudates. This project will focus on the PGPR strain Bacillus amyloliquefaciens SQR9, which was isolated from cucumber rhizosphere by this laboratory and applied widely in agricultural production as the microbial fertilizer. Previous studies showed that SQR9 revealed positive chemotactic response to several small molecular weight organic acids in cucumber root exudates. In this study, more chemotactic attractants of SQR9 in cucumber root exudates will be screened; then chemotactic analysis of mutants/complements and isothermal titration calorimetry (ITC) will be performed for identifying the methyl-accepting chemotaxis proteins (MCPs) in SQR9 for sensing these relevant ligands (including small molecular weight organic acids mentioned above). Finally, this proposal will also illustrate the molecular interaction characteristics between corresponding chemotactic attractant and MCP by competitive inhibition experiments, analytical ultracentrifugation, and differential scanning calorimetry. This study will contribute to understanding of molecular interaction mechanisms between plants and PGPRs, and to provide theoretical instructions for development and application of relevant microbial fertilizer products.

植物根际促生菌（PGPR）具有促生及生防功能，合理施用含PGPR的生物肥料是实现我国化肥减施、促进农业可持续发展的有效途径。生物肥料中的PGPR施入土壤后须在作物根际有效定殖才能发挥作用，而由根系分泌物介导的PGPR向根表的趋化反应是其有效定殖的重要前提。SQR9是本实验室分离自黄瓜根际并广泛应用的具有促生与生防功能的解淀粉芽孢杆菌，前期研究发现SQR9对黄瓜根系分泌物中的多种小分子有机酸表现出较强趋化性。本研究拟在此基础上继续筛选分泌物中诱导SQR9趋化反应的主要趋化物，并通过突变体/互补菌株趋化实验及等温滴定量热鉴定SQR9中负责感受相应趋化物（含上述小分子有机酸）的受体蛋白，最后通过竞争性抑制结合实验、分析性超速离心、差示量热扫描等生化方法阐明趋化物与受体蛋白之间的互作特征。本研究有助于深入理解PGPR与植物根系互作的分子机制，并为合理开发及施用相应的微生物肥料产品提供理论依据。

施用含有植物根际促生菌（PGPR）的生物肥料是实现我国化肥减施、促进农业可持续发展的有效途径，阐明促生菌的根际定殖机制可为提高肥料产品的应用效果提供理论依据。本项目以一株实验室广泛应用的优良PGPR菌株——解淀粉芽孢杆菌SQR9为对象，系统研究其趋化黄瓜根际的生物学机制，主要研究结果如下：1）构建菌株SQR9中8个趋化受体蛋白基因的全敲除突变体SQR9Δ8mcp，发现其根际定殖能力和对黄瓜根系分泌物的趋化能力显著弱于野生型；通过微流控系统趋化试验从根系分泌物中鉴定到菌株SQR9的39种趋化物（包括20种氨基酸、11种有机酸和8种糖/糖醇）和5种趋避物；通过回补菌株的趋化表型和分子结合试验发现McpA是可感受包括氨基酸（赖氨酸等）、有机酸（苹果酸等）和糖类（甘露糖等）等20种物质的多功能受体蛋白，McpC是主要负责感受氨基酸的受体蛋白，McpB负责感受4种氨基酸和2种有机酸，McpR是解淀粉芽孢杆菌特有的趋化受体，专门负责感受精氨酸；TlpA和TlpB的配体也被成功鉴定，而两种胞内的受体蛋白HemAT和YfmS则不参与菌株对根系分泌物的趋化。2）McpA和McpC是介导菌株SQR9趋化黄瓜根际和根表定殖的关键受体蛋白；利用靶标代谢组学定量检测了根系分泌物中能被McpA和McpC直接感受的20种物质的浓度，通过人工标准品复配的微流控趋化试验证明菌株SQR9对分泌物的趋化性主要由苹果酸、延胡索酸、葡萄糖酸、甘油酸、丙氨酸、赖氨酸、丝氨酸和甘露糖等主要趋化物决定；即根际促生菌的根际趋化是由少数主要趋化物和关键趋化受体主导的主效效应而非所有趋化物的累加效应决定。3）接种菌株SQR9可诱导黄瓜根部分泌更多的半乳糖，半乳糖不仅是SQR9的强趋化物（被McpA识别），还能通过McpA介导的方式促进菌株生物膜形成，从而增强菌株SQR9的根际定殖能力。综上所述，项目研究深入揭示了根际促生菌趋化作物根际的生物学机制，也为通过添加趋化物或定向改造促生菌等而增强生物肥料应用效果提供了思路和理论基础。