玫烟色棒束孢诱导的小菜蛾免疫相关基因鉴定及功能研究

The low virulence or failure of insect pathogenic fungi might be because of the insect immune defense system. Plutella xylostella is susceptible to entomopathogenic fungus Isaria fumosorosea, but the defense responses in P.xylostella is an important barrier against infection. The cascade of innate immunity in P. xylostella infected by I. fumosorosea is regulated by a set of immune genes and its molecular regulation network, but the immune-related genes and innate immune system of P. xylostella against I. fumosorosea remain largely unknown. In order to explore the immune-related genes and immune recognition regulatory network of P. xylostella, larvae will be inoculated with I. fumosorosea. We will use infected and non-infected larval of P. xylostella using the direct high-throughput paired-end RNA-sequencing, and quantitative proteomic analysis technique, combined with the analysis of genes association and differential expression profile. The expression level of immune-related genes at differential infected period, developmental stage and tissues of P. xylostella will be examined by means of real-time quantitative-Polymerase Chain Reaction detecting system. Moreover, we will verify the function of immune genes, the molecular regulation network, and the interactive mechanisms among the genes by using a specific RNA interference system of P. xylostella. The research results will illuminate the molecular basis and regulation mechanism on the innate immune mechanism of P. xylostella against I. fumosorosea. The findings generated from this study will provide an important significance both in theory and practice to promote the development and sustainable application of novel fungal bio-pesticides.

昆虫免疫系统对昆虫病原菌的抵抗，是延缓病原真菌杀虫速率的主要原因。利用昆虫病原真菌玫烟色棒束孢防治小菜蛾时，来自虫体的免疫抵抗作用是影响其防效的一个重要因素。小菜蛾防御玫烟色棒束孢是一个复杂的免疫基因表达网络调控过程，目前对这个免疫应答系统了解还相当欠缺，许多重要的免疫相关基因有待进一步探明。本研究拟对玫烟色棒束孢侵染前后的小菜蛾进行转录组测序、蛋白质组测序和蛋白表达差异谱研究，通过转录组和蛋白质组基因关联分析，筛选与玫烟色棒束孢侵染相关的免疫应答基因；利用实时荧光定量PCR技术分析免疫相关基因在不同感菌时期、虫龄和组织中的表达量差异；并利用RNA干扰技术验证特异性表达差异基因与免疫的关系，明确小菜蛾对玫烟色棒束孢免疫应答基因的功能及互作。研究结果对阐明小菜蛾对玫烟色棒束孢的免疫机制及其调控网络、新型高效真菌杀虫剂的开发与可持续应用均具有重要的理论和实践意义。

昆虫免疫系统对昆虫病原菌的抵抗，是延缓病原真菌杀虫速率的主要原因。小菜蛾防御玫烟色棒束孢是一个复杂的免疫基因表达网络调控过程，目前对这个免疫应答系统了解还相当欠缺，许多重要的免疫相关基因有待进一步探明。本研究通过玫烟色棒束孢侵染前后的小菜蛾转录组测序、蛋白质组测序和蛋白表达差异谱研究，获得1210个显著差异基因和285个显著差异蛋白，GO和KEGG分析表明这些差异基因和蛋白涉及解毒代谢、信号转导、生理防御和能量代谢等功能；通过转录组和蛋白质组关联分析，获得24个共同显著变化基因/蛋白，根据注释信息筛选出差异显著且上调表达的基因Cyp4v2和ZFPEP作为免疫关键候选基因。对Cyp4v2和ZFPEP的实时荧光定量PCR结果表明，两个基因时空表达模式差异较大，Cyp4v2基因在感菌24-32h间、3龄幼虫、血淋巴和脂肪体中表达量最高，在小菜蛾抵御玫烟色棒束孢免疫应答中起重要作用；建立起针对小菜蛾免疫关键基因Cyp4v2的RNAi体系，沉默Cyp4v2基因能有效促进玫烟色棒束孢对小菜蛾的致病作用，使幼虫免疫能力（血细胞总数、酚氧化酶和溶菌酶活性）显著降低，证明Cyp4v2基因功能与小菜蛾对玫烟色棒束孢免疫防御有关。本研究结果为阐明小菜蛾对玫烟色棒束孢的免疫机制及其调控网络提供了研究基础和理论依据。