绿针假单胞菌SPS-41产挥发性物质杀线虫作用机理的研究

Plant parasitic nematodes (PPNs) cause huge economic losses in agriculture and forestry every year，and the research of nematicidal VOCs produced by microorganisms has attracted more attention. At present, most of the studies on the mechanism of killing nematodes take the model organism C. elegans as the research object, but the mechanism of VOCs killing nematode has not yet been clarified. A biocontrol strain, Pseudomonas chlororaphis SPS-41, has been isolated from the root of plants in our previous stage. It produce nematocidal volatile substance, and induce the expression of gene daf-16 in nuclear, which play critical role in nematode immunity, but the mechanism is unclear. In this study, we proposed to find the target of volatile substances based on the experimental methods such as RNA interference, screening mutation library and gene rescue, to elucidate the olfactory signal transduction pathway of nematode sensing volatile substances. Combined with the response induced by volatile substances, researching the molecular mechanism of the activation of the immune signal pathway mediated by olfactory signal based on the methods of GFP markers, nematodes hybridization and so on, to clarify the mechanism of immune response mediated by olfactory. The research result is great scientific significance to reveal the mechanism of VOCs killing nematode, and the interaction between nematode and microorganism, and it provide a new opportunity and scientific theoretical basis for the research of biocontrol agent against plant parasitic nematode.

植物寄生线虫每年给农业、林业造成巨大经济损失，微生物产生挥发性有机物（VOCs）杀线虫已成为研发线虫生防制剂的热点领域之一。目前对杀线机理的研究多以模式生物秀丽隐杆线虫为研究对象，然而对生防菌VOCs杀线机理尚未阐明。申请人前期从植物根际分离筛选到一株高活性杀线菌株绿针假单胞菌SPS-41，可通过产生挥发性物质毒杀线虫并诱导线虫免疫关键基因daf-16的入核表达，但其作用机理并不清楚。本研究拟通过线虫RNA干扰、突变文库筛选以及基因回补等方法，寻找挥发性物质作用靶点，阐明线虫感知VOCs的嗅觉信号传导途径；结合VOCs诱发的免疫应答，通过GFP标记、线虫杂交等方法，阐明线虫嗅觉介导的免疫信号途径激活的分子机理。研究结果对于揭示高效生防菌株SPS-41的VOCs杀线虫机理、线虫与微生物的相互作用关系具有重要科学意义，并为植物寄生线虫生防技术研究提供新的切入点与科学理论依据。

在自然环境中，细菌作为最大的功能种群之一，在维持生态系统平衡中发挥了重要的作用，但目前人们对细菌潜在功能的开发还初于初步阶段。秀丽隐杆线虫作为研究生物发育、程序性死亡、神经信号传导的优良模式生物，其遗传背景已被深度解析，因其研究优势，其也成为筛选毒杀病原线虫、研究毒力因子作用方式的优良模式生物。我们前期的研究中，筛选获得了一株根际促生细菌，经生理生化、分子鉴定，该菌为绿针假单胞菌。我们发见这株菌对线虫有极强的毒杀活性，通过分析菌株SPS-41毒杀线虫的不同方式，我们进一步研究了其产生的挥发性有机物质对线虫的毒杀机理。菌株SPS-41可产生多种挥发性物质毒杀线虫，其中辛酸乙酯对线虫的毒杀作用最强。通过转录组分析了线虫对于菌株SPS-41产生的挥发性物质的应答基因及相关途径，发现其氧化还原途径相关基因显示出明显的表达差异性，尤其是P450相关基因，通过KEGG信号通路分析，p450解毒途径相关基因转录水平显示出明显的差异性。我们同时筛选了一些化感神经相关的基因，发现在G蛋白α亚基ODR-3和GPA-3双突变的线虫，其在挥发物作用后，其存活率比野生型线虫有明显的上升，其他部分化感基因对于线虫感知挥发性物质也有明显的影响，确定了线虫通过化感神经元感知挥发性物质，进而激活了P450解毒途径。