蜡蚧菌在侵染西花蓟马过程中表面蛋白的功能分析及其植物内生性的作用

Frankliniella occidentalis, a very important invasive pest, is becoming more and more serious because of its strong adaptability. Lecanicillium spp. are the most important pathogenic fungi and have pathogenicity to F. occidentalis. In order to determine the function of surface proteins of L. spp. and its role as endophytic fungal entomopathogen, the present proposed project will focus on following aspects. The types of surface proteins of spores just before and after L. spp. contacting F. occidentalis will be determined by comparative proteomics analysis. The major proteins will be predicted and selected by the method of bioinformatics. The relationship of surface proteins with pathogenicity to F. occidentalis will be explored by using green fluorescence protein(GFP), gene knockout and overexpression technology, and relationship of surface proteins with the immune responses in F. occidentalis will be revealed too.The target proteins in F. occidentalis acted by surface proteins will be assessed by co-immunoprecipitation and yeast two-hybrid technology. Endophytic colonization of L. spp. in kidney bean will be investigated by GFP. The changes of activities of protective enzymes, detoxifying enzymes and digestive enzymes in F. occidentalis, and the development and fecundity of F. occidentalis will be assessed by population ecology and biochemical analysis. The results will lay a foundation for further exploring pathogenic mechanism of L. spp. to F. occidentalis, and providing theory basis for tritrophic interactions of L. spp. , bean and F. occidentalis.

入侵害虫西花蓟马由于强大的适应性，为害不断加重。蜡蚧菌是重要的病原真菌，对西花蓟马有一定的致病性。为深入了解蜡蚧菌表面蛋白在侵染过程中的作用及蜡蚧菌植物内生性。本项目通过比较蛋白组学分析蜡蚧菌与西花蓟马接触前后孢子表面蛋白的种类，并通过生物信息学方法，预测和筛选起主要作用的蛋白质；通过绿色荧光标记，并利用基因敲除、超量表达等技术，揭示蜡蚧菌孢子表面蛋白与西花蓟马致病性及与西花蓟马体内免疫反应之间的关系；通过免疫共沉淀和酵母双杂交技术明确表面蛋白在西花蓟马体内的作用靶标蛋白。通过绿色荧光标记探究蜡蚧菌在菜豆中的定殖情况；运用种群生态学和生化方法，研究蜡蚧菌定殖后的菜豆对对西花蓟马生长发育、繁殖和体内保护酶、解毒酶和消化酶活性的影响。研究结果将为进一步揭示蜡蚧菌对西花蓟马的致病机理奠定基础，为明确蜡蚧菌-菜豆-西花蓟马的互作关系提供理论依据。

西花蓟马Frankliniella occidentalis对全世界蔬菜花卉等经济作物造成了极大的危害。蜡蚧菌是重要的病原真菌，在害虫生物防治中占有重要的地位。本项目开展了蜡蚧菌对西花蓟马的致病性及致病机理、蜡蚧菌在菜豆植株中的内生性及其作用等研究。. 通过多基因位点对目前已知的蜡蚧菌所有物种进行了系统发育学分析。选用7株蜡蚧菌菌株，研究了它们对西花蓟马蓟马的致病性，发现蛀虫蜡蚧菌和渐狭蜡蚧菌对西花蓟马有较高的致病力，对两种蜡蚧菌的培养条件进行了优化。渐狭蜡蚧菌和蛀虫蜡蚧菌侵染西花蓟马后，除造成蓟马直接死亡外，还使西花蓟马发育时间显著缩短，内禀增长率、周限增长率和净繁殖率都明显降低, 存在明显的亚致死效应。另外蓟马体内保护酶和解毒酶活性，体内激素含量，体内微生物组成及比例均发生了明显的变化，且两种蜡蚧菌造成的影响不同。完成了蛀虫蜡蚧菌和渐狭蜡蚧菌全基因组的测序及数据组装，推测可能提高对寄主毒力的蛋白有5种。成功构建了蜡蚧菌遗传转化体系及明确了重要蛋白基因的功能。蜡蚧菌的内生性对菜豆植株有一定的促生作用，但对西花蓟马的生长发育、繁殖、种群参数及体内酶活性有明显的影响。渐狭蜡蚧菌造成西花蓟马卵巢发育迟缓且提前萎缩，对卵黄原蛋白基因表达量及含量有明显的影响。研究结果为西花蓟马的绿色治理及拓展虫生真菌防治害虫的途径奠定了基础。