小菜蛾肠道共生菌介导Toll信号通路对Bt抗性的分子机制

The diamondback moth, Plutella xylostella (L.), is a worldwide pest with highly risk of insecticide resistance. It is not only resistant to various chemical insecticides, but also the first report of insect resistant to Bacillus thuringiensis (Bt) in the field. Our previous studies showed that the gut symbiotic bacteria of Enterobacter sp.IAE5 enhanced the P. xylostella resistance to Bt, and the Toll pathway in immune system of diamondback moth may be involved in the process. However, the exact molecular mechanism is still unknown. In this project, the Western blot, ELISA and Immunohistochemistry and other experimental techniques will be conducted to identify the pattern recognition receptors of the midgut immunity which recognize the Enterobacter sp.IAE5; the Co-IP, Dual-luciferase reporter assay system and Antibody-blocking were used to determine the Toll signal pathway which response to the Enterobacter sp.IAE5; the RNAi and Immune priming were used to confirm the effect of Toll pathway in the resistance of P. xylostella to Bt. Our study will elucidate the mechanism of Bt resistance in P. xylostella mediated by gut symbiotic bacteria. It is of great significance of the project to reveal the mechanism of gut symbiotic bacteria mediated insecticide resistance, and can also provide a theoretical basis for the resistance management and sustainable control of the P. xylostella.

小菜蛾是抗药性极强的害虫，不仅对多种化学杀虫剂产生抗性，也是第一个报道在田间产生对Bt抗性的昆虫。我们前期研究发现肠道共生菌Enterobacter sp.IAE5能够提高小菜蛾对Bt的抗性，且小菜蛾免疫系统Toll通路参与了该过程，但具体的分子机制并不清楚。本项目拟通过Western blot、ELISA和免疫组化等技术，鉴定小菜蛾中肠识别共生菌Enterobacter sp.IAE5的病原模式识别受体；利用Co-IP、双萤光素酶报告基因系统和体内抗体阻断等技术确定小菜蛾中肠响应Enterobacter sp.IAE5的Toll信号通路；使用RNAi和免疫致敏等技术明确Toll信号通路在小菜蛾对Bt抗性中的作用，阐明肠道共生菌介导宿主免疫影响Bt杀虫活性的效应和机制。本项目对揭示肠道共生菌介导的杀虫剂抗性具有重要意义，同时也可以为小菜蛾的抗性治理和持续控制提供理论基础。

肠道微生物通过与宿主昆虫互作，在宿主的营养供给、生长发育、代谢物解毒、病原体防御和农药抗性等方面发挥重要作用。随着“宿主-肠道微生物互作”研究领域的不断扩大，越来越多的研究表明昆虫肠道微生物与苏云金芽胞杆菌（Bacillus thuringiensis, Bt）抗性密切相关。小菜蛾Plutella xylostella是对十字花科作物具有极强破坏性的世界性害虫之一，同时又是田间最早产生Bt抗性的害虫，已成为研究Bt抗性最主要的模式生物，其肠道微生物与Bt抗性之间的关系值得探讨。本项目通过深入研究小菜蛾肠道微生物与Bt抗性之间的关系，发现了小菜蛾肠道微生物能够介导宿主对Bt的抗性，其抗性机制包括定殖抗性、保护宿主肠道内壁以抑制和减缓Bt毒素对肠道的破坏、以及通过激活宿主的免疫信号通路Toll通路，激活下游抗菌肽等效应因子的表达，促进机体对Bt菌株的抗性。研究表明小菜蛾肠道微生物在宿主对抗Bt过程中的作用是多方面的，也进一步提示我们在研究生物体的互作及其功能的时候，需要从更加综合的视角来看待科学问题。本项目通过系统的研究确定了小菜蛾肠道微生物介导宿主免疫Toll信号通路上的关键基因、互作、及功能，明确了“小菜蛾肠道微生物-宿主免疫-Bt抗性”之间的关联，解析了小菜蛾肠道微生物介导宿主对Bt抗性的分子机制，全面和丰富了小菜蛾抗性机理的研究，为小菜蛾的抗性治理和持续控制提供理论基础。