家蚕对白僵菌感染免疫应答相关基因研究

Beauveria bassiana is an important pathogenic fungus to silkworm, an economic insect, and also one of fungal insecticides commonly used in controlling agricultural and forest insects. Up to data, most of the antimicrobial peptides identified so far are not effective to fungi, and the defense mechanism of silkworm and other insects to fungal infection remains unclear. The approach of digital gene expression (DEG) profiling will be employed in the project to screen and identify differentially expressed genes of silkworm against B. bassiana infection. Then，expression characteristics and transcript regulation of the differential genes, tissue and cell localization and signal transduction pathways of the differential proteins will be explored. Inhibitory and killing function of the differential proteins to B. bassiana will also be studied. The function of differential genes will be further verified with RNA interference (RNAi). Based on the results of the researches, the responsive mechanism of silkworm against B. bassiana infection will be speculated. Hopefully, the results obtained in the project will provide new theoretical basis for understanding at a molecular level the defense mechanism of silkworm against fungal infection and the pathogenic mechanism of B. bassiana to the silkworm and for controlling the B. bassiana caused disease in sericultural production. Meanwhile, the results will also be good reference to understanding the molecular pathogenic mechanism of other entomopathogenic fungi.

白僵菌是家蚕重要的病原真菌，也是害虫生物防治中常用的真菌性杀虫剂之一。目前，在昆虫中发现的抗菌肽绝大部分对真菌无明显的作用，对于家蚕和其他昆虫防御真菌的分子机制尚不清楚。本课题拟运用数字基因表达谱技术，构建家蚕感染白僵菌后差异基因表达谱，筛选获得家蚕感染白僵菌的差异表达基因，通过对差异表达基因的表达特征、转录调控、差异蛋白的组织细胞定位、差异基因表达产物的细胞信号转导通路、差异基因表达产物对白僵菌的抑杀作用以及差异表达基因的RNA干涉（RNAi）对家蚕感染白僵菌发病过程的影响等研究，解析家蚕对白僵菌感染免疫应答反应的分子机理。为从分子水平上阐明家蚕对白僵菌感染的防御机制和白僵菌对家蚕的致病机理提供新的理论依据，并为家蚕僵病防治提供新的研究方向。同时，为阐明其他类型昆虫真菌致病的分子机制提供有益的参考。

白僵菌是家蚕重要的病原真菌，也是害虫生物防治中常用的真菌性杀虫剂之一，目前对于家蚕及其他昆虫防御真菌的分子机制尚不清楚。本项目构建了家蚕幼虫被白僵菌感染后的差异表达基因文库，筛选获得1430个差异表达基因，并在家蚕感染白僵菌后的血淋巴中筛选获得30个差异表达蛋白；GO和KEGG分析表明这些差异表达基因和蛋白涉及家蚕防御应激、信号转导、吞噬作用等相关的生物功能。对差异表达基因的实时定量分析结果表明，白僵菌侵染家蚕之后，能激活家蚕Toll信号通路、Imd信号通路、Jak/STAT信号通路和黑化作用相关基因的表达，各信号因子在感染的不同时期表达水平差异显著；10种Bmserpins的表达模式差异显著，酚氧化酶活化级联反应相关基因在白僵菌侵染家蚕的整个过程中都显著上调表达，说明白僵菌侵染家蚕会引发强烈的黑化反应，黑化作用在昆虫抗真菌免疫中起到重要作用；Toll和Jak/STAT信号通路的RNAi干涉以及信号通路特异性抑制剂能抑制信号传递，且导致家蚕血淋巴抗真菌活性和对白僵菌感染抵抗性的降低。家蚕感染白僵菌后，10余种抗菌蛋白基因均不同程度的上调表达，用原核表达系统表达了家蚕抗菌肽等抗菌相关蛋白，但只有BmCecA、BmCecB和BmAttacin三种抗菌肽对白僵菌有明显的抑菌作用，BmGlv2单独对白僵菌没有抑菌作用，但却具有与BmCecA的协同抑菌作用，能增强BmCecA对白僵菌的抑菌作用。家蚕体内注射BmCecA能有效抑制白僵菌对家蚕的致病作用，延长被感染蚕的发病死亡时间。家蚕感染白僵菌后，脂肪体、中肠、血淋巴中GSTs、GR、GSH-Px酶活力显著提高，其相应基因的表达量也显著上调表达，表明家蚕幼虫GSTs和GSH-Px构成的解毒和抗氧化防御系统在抗真菌感染过程中起到重要作用。上述研究结果为从分子水平上阐明家蚕对白僵菌感染的防御机制和白僵菌对家蚕的致病机理提供了新的理论依据。