蜕皮液介导的昆虫体壁免疫机理研究

The application of chemical pesticides brings damage to human health. Thus, people are paying much more attention to the non-pollution biological pesticides. The entomopathogenic fungi Beauveria bassiana is a type of bio-pesticide. However, the application of Beauveria bassiana is limited due to its capacity of evading the host innate immunity. Our previous published paper demonstrates that the silkworm Bombyx mori are very effective to fight against Beauveria bassiana infection in unknown mechanism during the molting stages. Preliminary experiments demonstrate that when the molting fluids were removed by washing after ecdysis, the insects show low resistance to Beauveria bassiana infection, which indicates that there are inhibition factors in the molting fluids. Further works show that prophenoloxidase and some other proteins in the molting fluids including purified prophenoloxidase can bind to the spores of Beauveria bassiana. To our surprise, prophenoloxidase was activated to show enzyme activity if it was bound to the spores. Very interestingly, proteinase was secreted by spores of Beauveria bassiana to degrade free prophenoloxidase in the solution. In this project, we work on the molecular mechanism of inhibition factors on Beauveria bassiana infection derived from the molting fluids, by which to disclose the special epidermis immunity response during ecdysis. In the future, these inhibition factors may be targets in the fields of pest biological control or economical insect production. We believe that this work is important due to the important scientific problems and potential agricultural application.

化学农药的使用给人类健康带来了危害，人们对无污染生物农药的使用日益重视，昆虫致病真菌白僵菌是一种具有潜力的生物农药，但是其主动逃避宿主免疫的能力制约了推广和应用。前期发表的结果表明在蜕皮期间的家蚕体壁（包括新旧表皮和蜕皮液）免疫可以有效防御球孢白僵菌感染，但其分子机制不清楚。预实验显示在蜕皮后蜕皮液若被水清洗，家蚕幼虫对白僵菌感染抵抗力下降，表明蜕皮液中存在抑制白僵菌侵染的因子。初步研究还发现蜕皮液多酚氧化酶等多种蛋白可以结合到白僵菌分生孢子上，纯化的多酚氧化酶结合到孢子后被激活，而白僵菌分生孢子又可以分泌未知蛋白酶来剪切游离的多酚氧化酶，和孢子结合的蜕皮液蛋白对白僵菌侵染的抑制功能有待于深入研究。本项目拟解析蜕皮液中抑制白僵菌侵染的因子，以期揭示眠期昆虫有效对抗真菌的分子机理。昆虫蜕皮液抑制因子有可能作为新型靶标，为害虫生物防治和益虫保护提供参考作用，具有重要的科学研究和应用价值。

昆虫上皮组织（皮肤）是保护昆虫免于病原菌感染的第一道防线，其上皮细胞分泌许多蛋白构成角质层，保护昆虫不受机械损伤和水分的过度蒸发。昆虫的皮肤组织也存在免疫反应，对其免疫机制的研究有助于提高农业益虫的皮肤保护功能，同时也为通过破坏皮肤免疫进行的农业害虫生物防治提供靶标基因。与哺乳动物不同，昆虫在生长发育过程中具有周期性蜕皮的现象。刚刚蜕皮的昆虫新表皮还没有完全硬化，但是能够成功避开病原体的侵染，其中的机制仍然不清楚。昆虫在蜕皮期间，在新旧表皮之间会产生蜕皮液，蜕皮液一直被认为是蜕皮时用于产生机械压力，从而帮助新旧表皮的分离。针对这一现象，收集幼虫的蜕皮液，通过蛋白质质谱鉴定，发现蜕皮液存在许多蛋白，包括免疫蛋白，进一步对蜕皮液中的多酚氧化酶免疫保护功能进行研究，发现蜕皮液残可以留在新表皮上，蜕皮液多酚氧化酶与白僵菌孢子结合后能够提高白僵菌孢子的疏水性，从而降低了孢子与昆虫皮肤的粘附性，并因此大大减弱白僵菌孢子的侵染力。在白僵菌侵染的时候，昆虫劫持孢子分泌的蛋白酶BPS8，激活多酚氧化酶，产生的黑色素可以抑制孢子的萌发。由此可见，昆虫进化出了一套独特的体外免疫机制，利用新表皮中残留的蜕皮液多酚氧化酶来抵抗蜕皮后空气中致病菌的感染。相关研究对提高白僵菌作用效率具有重要的参考作用