虫生真菌鹿儿岛被毛孢对两种昆虫激素应答的分子机理

As a host-special entomopathogenic fungus, Hirsutella Pat is an important pathogenic factor of various arthropods and nematodes, with great value for biological control. Previous research has found that insect hormone can significantly promote the conidia mucilage secretion. It suggests that insect hormone is likely to be one of the main pressures factor during the process of fungus colonization in insect haemocoel, causing a series of response in fungus. However, it is not clear that the direct action relationship between entomogenous fungi and insect hormones, as well as the specific mechanism of the Hirsutella response to insect haemocoele. This study employs H. satumaensis Aoki, hydroxyecdysone and Juvenile hormone Ⅲ as materials, using the protein expression profile, comparative transcriptome technology and lection of conidia mucilage to reveal the molecular mechanism of Hirsutella response to insect hormone from protein transcription, functional gene expression and metabolic pathways several aspects. This study can further understand immune escape and colonization mechanism of pathogenic fungi in host, revealing the role of conidia mucilage in the environment adaption and in the process of infection. It will enrich and improve the interaction mechanism between insects and entomopathogenic fungi; providing a theoretical basis and application technology for the genetic improvement of insecticidal fungi on special hosts, high efficiency and non-resistant.

被毛孢（Hirsutella Pat.）属于窄谱类虫生真菌，是多种节肢动物和线虫的重要病原真菌，极具生防价值。前期研究发现，昆虫激素可显著促进其胞外粘液的分泌，说明激素可能是虫生真菌在昆虫体内定殖需响应的重要环境因子，可引起虫生真菌的系统应答。但两者之间的直接作用关系及应答昆虫激素的具体机制尚不清楚。研究拟用鹿儿岛被毛孢、蜕皮激素和保幼激素为材料，通过比较转录组学、蛋白表达谱技术、粘液中蛋白成分的鉴定及相关基因功能的验证等方法，从蛋白转录水平、基因表达和代谢通路几个层面研究该菌应答昆虫激素的分子机制，探索虫生真菌适应昆虫血体腔内环境机制的多样性。研究可进一步理解虫生真菌对寄主的免疫逃避与体内定殖机制，揭示胞外粘液在内环境适应和对寄主侵染过程中扮演的角色，丰富和完善昆虫与虫生真菌相互作用机理；为杀虫真菌的遗传改良及窄谱、高效和不易产生抗性的生物农药开发方面提供理论基础和应用技术。

项目根据昆虫激素能调节被毛孢分生孢子的胞外粘液分泌的现象入手，通过转录组和蛋白组联合分析证实了鹿儿岛被毛孢与昆虫蜕皮激素和保幼激素存在直接相互作用关系。结果发现分泌性小分子蛋白、昆虫激素代谢类基因和凝集素类蛋白等均参与应答过程，证明了对激素的应答是真菌在昆虫体腔定殖重要环节之一。对鹿儿岛被毛孢胞外粘液成分及对孢子表面性质的影响进行了研究。验证了粘液能影响孢子粘附和毒力，粘液成分与其功能联系紧密，粘液中含有中性糖、碱性糖和碱不溶性糖等糖类物质及凝集素类蛋白。说明粘液成分与其功能（黏附、识别等）联系紧密。构建了鹿儿岛被毛孢的遗传转化体系，并依据该转化体系对筛选出的重要应答基因—蜕皮激素失活酶Hsegt和凝集素类蛋白Hscal1的生物学功能进行了研究，探究了它们在响应昆虫激素过程中的作用方式。将昆虫激素作为虫生真菌在血体腔定殖时要面对的胁迫因子来探讨真菌与寄主昆虫的互作关系。从分子层面解析了鹿儿岛被毛孢应对昆虫蜕皮激素（20E）和保幼激素（JH）压力而产生的具体应答分子机理，发现鹿儿岛被毛孢应对昆虫激素胁迫存在特异性应对策略。研究结果表明，鹿儿岛被毛孢入侵寄主后，昆虫会通过上调20E浓度来增强免疫反应，抑制真菌的生长。相应地，真菌中存在昆虫激素代谢相关基因，如真菌可以通过上调20E失活酶降低20E浓度，进一步抑制昆虫免疫水平，使其达到快速增殖的目的。另外，进一步对鹿儿岛被毛孢中的重要应答基因蜕皮激素失活酶（Hsegt）和凝集素类蛋白Hscal1的生物学功能进行了深入研究。