白僵菌核糖体毒素蛋白Ribotoxin对昆虫天然免疫抑制机制研究

Utilization of chemical pesticides without limitation seriously threatens the human health and environmental ecology. Bio-pesticide is safe, and its utilization has been paid attention to for a long time. However, the insect innate immunity against microbes limits the efficiency of Bio-pesticides. Beauveria bassiana is a type of very important Bio-pesticide. Our preliminary works show that Ribotoxin can be secreted into hemolymph after Beauveria bassiana surficial infection. When Ribotoxin was knocked out, the loss-of-function mutant cannot inhibit prophenoloxidase-induced melanization in cuticle, the productions of some antibacterial proteins in fat bodies and Reactive Oxygen Species in hemocytes. These works demonstrate that the secretion of Ribotoxin by Beauveria bassiana can effectively inhibit the immunity responses in the hosts. However, the regulation mechanism is unclear. To this end, Beauveria bassiana species like wild type, loss-of-function of Ribotoxin and Ribotoxin complement will be utilized to infect insects through surface and injection respectively. After that, immunity responses in epidermis, fat bodies and hemocytes will be compared. In the meantime, spores of the above three species to bind with prophenoloxidase and activate it will be compared to understand the melanization of Ribotoxin mutant in cuticle. Based on the concentration of Ribotoxin in the host hemolymph after being secreted by Beauveria bassiana hyphae, the purified recombinant Ribotoxin will be injected into larvae with pathogen-associated molecular pattern from fungi and bacteria, beads and bacteria. And then detection of pathogens, expression of immunity genes, hemocyte phagocytosis, encapsulation, cell proliferation and death will be studied. With these studies, it is likely helpful to understand the inhibition on host immunity by Ribotoxin, which may be utilized for pest biological control as a tool.

化学农药使用不可避免危害人类健康和生态安全，无公害生物农药使用受到日益重视，但昆虫天然免疫反应制约了生物农药的作用效率。前期通过生物农药白僵菌体表感染昆虫，发现核糖体毒素蛋白被分泌到宿主血清中；敲除该基因，突变体对角质层多酚氧化酶介导的黑化反应、脂肪体部分免疫基因表达和血细胞活性氧产生均不能像野生型一样有效控制，反向证明该蛋白以未知机制抑制宿主免疫。为此，利用野生型、敲除突变和回补等分别进行体表和伤口感染，系统研究核糖体毒素蛋白对宿主不同组织免疫活性影响，同时比较其介导孢子和多酚氧化酶结合、激活和对黑化反应的不同；以宿主血清中该蛋白浓度为基准，将纯化的蛋白分别和病原表面识别化合物、细菌等混合注射，确定其是否通过影响病原物识别、免疫基因表达、细胞功能等机制来抑制宿主免疫反应。综合研究结果，分析核糖体毒素蛋白对宿主免疫抑制的分子通路，并加以验证，为基因工程改造生物农药来增强作用效率提供参考。

化学农药使用不可避免危害人类健康和破坏生态安全，无公害生物农药应用受到日益重视，但是昆虫天然免疫反应制约了生物农药作用效率，因此获得有效抑制宿主免疫功能的基因，加以有效利用，是提高生物农药重要途径。白僵菌是重要的生物农药，在孢子发芽时白僵菌核糖体毒素蛋白（Ribotoxin）被分泌到宿主血清中，通过构建Ribotoxin的敲除和回补品系，研究发现突变体对表皮多酚氧化酶介导的黑化反应、脂肪体免疫基因表达和血细胞活性氧均显著下降，证明核糖体毒素蛋白核糖体毒素蛋白对宿主免疫具有抑制作用。进一步通过构建过表达核糖体毒素基因品系，生测表明可以显著增强杀虫效率，有望为增强白僵菌生物农药作用效率提供一个重要的工具基因。相关研究具有重要的科研和应用价值。