吸引线虫的细菌多样性及其吸引分子机制研究

Volatile organic compounds (VOCs)-mediated attraction of nematode by bacteria is one of the important contents on the interactions between bacteria and nematode, and it has potential application in biocontrol of plant-parasitic nematodes. This project focuses on nematode-attracting bacteria and their bioactive VOCs, to characterize the biodiversity of bacteria producing nematode-attracting VOCs by screening, identification and phylogenetic analysis taking the Meloidogyne incognita and Caenorhabditis elegans as targets; to reveal the material basis for VOCs-mediated interactions between bacteria and nematode by using of the SPME-GC/MS analysis and confirmation with individual commercial compounds to identify the main components of nematode-attracting VOCs; to screening and determination the main olfactive chemotaxis neurons, Gα and downstream signal transduction pathways to senese the attractive signal molecule in C.elegans, using the molecular biological methods such as gene function-lost mutant, RNA interference, cell immunofluorescence taking C. elegans as target and the representive nematode-attracting VOCs as signal molecules. Results of our project will provide rich resource and the scientific basis for the application of nematode-attracting bacteria and their bioactive VOCs in biocontrol of nematode.

挥发物介导细菌吸引线虫是细菌与线虫互作中的重要内容之一，在线虫生防领域具有潜在的应用价值。本项目围绕细菌及其活性挥发物吸引线虫作用的研究主题，以南方根结线虫（Meloidogyne incognita）和秀丽隐杆线虫（Caenorhabditis elegans）为靶标，通过资源筛选、鉴定和系统发育分析，阐明挥发物吸引线虫的细菌物种多样性特征；通过SPME-GC/MS分析和纯品验证方法鉴定细菌吸引线虫的主要挥发物组分，揭示吸引性挥发物介导的细菌-线虫互作的物质基础；以秀丽隐杆线虫为靶标，以代表性吸引挥发物为信号分子，通过基因突变、RNAi、细胞免疫荧光检测及突变回复和显微注射等分子生物学手段明确线虫感知细菌挥发物吸引的嗅觉趋化神经元、G蛋白α亚基及下游信号转导途径，初步阐明细菌及其挥发物吸引线虫的分子机制。本项研究结果将对吸引线虫的细菌及其挥发物在线虫生防中提供丰富资源及应用的科学依据。

本课题以南方根结线虫（Meloidogyne incognita）和秀丽隐杆线虫（Caenorhabditis elegans）为靶标，，从不同类型生境和基质中分离筛选和识别了吸引线虫的细菌群体，并特征性分析其群体多样性，，基于16SrRNA 基因序列系统发育分析，阐明挥发物吸引线虫的细菌种群组成特征及特定生态位的分布特征，这154株吸引线虫的细菌分别属于放线菌门（Actinobacteria）、厚壁菌门（Firmicutes）和变形菌门（Proteobacteria）中的28个属，链霉菌属（Streptomyces）是群体中的优势属。应用过SPME-GC/MS 相结合的方法和纯品验证方法分析了细菌吸引线虫的挥发物质谱组分，识别验证了南方根结线虫和秀丽隐杆线虫共同吸引的由线虫吸引细菌产生的主要挥发性酮类物质。随后，采用适应性和优选实验判定了引发线虫产生吸引性行为的重要酮类挥发物质成分，揭示吸引性酮类挥发物介导的细菌-线虫互作的气味物质基础；以秀丽隐杆线虫为靶标，以吸引性酮类挥发物为信号分子，通过行为学和遗传学手段明确线虫感知线虫吸引性细菌产生的酮类挥发物吸引作用的嗅觉趋化神经元、G 蛋白α 亚基及下游信号转导途径。本项目研究结果为解析细菌如何通过其释放的挥发物吸引线虫提供了良好的研究模型，增加了我们对在自然界线虫与细菌互作的深入了解，为增加线虫生防有效性提供了可用的信息。