贵阳腐霉菌CRN类效应子的杀蚊功能与作用机制研究

贵阳腐霉（Pythium guiyangense Su）是由我国科学家分离出来的一株具有灭蚊活性的生防菌，隶属于卵菌。已知卵菌多数为植物病原菌，通过产生效应子来干扰寄主免疫反应甚至杀死细胞，但关于昆虫生防卵菌效应子的功能与机制目前还知之甚少。本项目拟选取贵阳腐霉代表性CRN效应子，利用遗传转化方法逐一评估它们对蚊虫的杀虫活性，获得致病关键CRN效应子；表达纯化后，研究它们对蚊虫的体外毒力效应，获得新型杀虫蛋白；利用酵母双杂交方法在蚊虫中筛选其靶标蛋白，进一步研究靶标蛋白在蚊虫免疫反应中的功能，阐明效应子与其互作干扰其正常功能的机制。同时与其它植物寄生腐霉菌的CRN效应子比较，探讨腐霉菌适应不同寄主的分子进化机制；本研究将揭示CRN效应子在贵阳腐霉侵染蚊虫过程中的功能与机制，明确贵阳腐霉的致病机理；阐述腐霉菌侵染不同寄主（昆虫与植物）的适应机制；还有望为害虫控制提供有价值的新基因和新蛋白。

Pythium guiyangense Su is an oomycete pathogen of mosquitoes isolated from mosquito larvae by a Chinese scientist and has been proved a promising agent for biological control of mosquitoes. Most of oomycetes are plant pathogens which could manipulate host immune responses or even kill host cells by producing a large number of effector proteins with different biochemical functions. P. guiyangense is also a member of oomycetes, however the biochemical activities of effector proteins produced by P. guiyangense to mosquitoes and molecular mechanisms are still not clear. In this project, several CRN effector proteins of P. guiyangense will be chosen to evaluate their infection to mosquitoes using genetic transformation methods. The virulent effector proteins during mosquito infection will be expressed using prokaryotic and eukaryotic expression systems. The obtained proteins will be purified for testing infection rates to mosquitoes。Meanwhile, target proteins of CRN effector proteins in mosquitoes will be investigated using Yeast Two-Hybrid systems, followed by exploring the molecular mechanisms of interactions between CRN effectors and target proteins. Additionally, comparing with CRN effectors from plant-pathogenic Pythium species, the evolutionary mechanism of Pythium species with different hosts will be investigated. This project will uncover the virulent function and molecular mechanisms of P.guiyangense CRN effectors in the evolutionary adaptation to the mosquito host, and will be expected to provide valuable genes for controlling insect pests.