小菜蛾免疫防御虫生真菌侵染的分子机制

Immunity defense of insect against fungal infection is a complex process, which involved in the dynamic strategy of immune gene expression. however, its regulation mechanism is not clear. In this project, we intend to use Plutella xylostella as the research object. On the basis of transcriptome and small RNA libraries has been sequenced, RNA-seq and Microarray will be used to screen the mRNAs(named F-mRs) and miRNAs(named F-miRs) which expressed in the different time of fungal infection. Quantitative RT-PCR and Northern blot will be used to verify the spatial and temporal expression profiles of other immune-related genes, after F-mRs and F-miRs are silenced by RNA interference (RNAi) to elucidate the functions of F-mRs and F-miRs in the immune process of p. xylostella against the infection of entomopathogenic fungi. The software will be used to predict gene target of F-miRs, and then confirmed by luciferase system and RNAi, the result will clarify the dynamic correlation of F-mRs and F-miRs in the immune process of P. xylostella. From the above research will elucidate the dynamic molecular mechanisms of the immunity system of P. xylostella against the infection of entomopathogenic fungi. The results of this project will not only guide to explore the new green fungal pesticides, but also lay a basis for biological control of insect pests based on the regulation of insect immune system.

昆虫免疫真菌的侵染，是一个复杂的分子动态过程，体现在免疫基因表达策略，但表达调控机制不清楚。本项目拟以小菜蛾为研究对象，在已测定小菜蛾转录组和小RNA文库的基础上，利用RNA-seq和基因芯片筛选小菜蛾免疫虫生真菌侵染不同时间的mRNAs(F-mRs)和microRNAs(F-miRs)，RNAi分别沉默F-mRs和F-miRs在小菜蛾体内的表达，运用定量RT-PCR、Northern blot检测小菜蛾免疫基因的表达差异，阐明F-mRs和 F-miRs在小菜蛾免疫真菌中的功能；利用荧光素酶载体系统和RNAi技术明确F-miRs调控的靶标基因，并明确F-mRs和F-miRs在免疫真菌中的动态关系。通过上述研究，阐明F-mRs和F-miRs调控小菜蛾免疫真菌侵染的分子机制。研究结果不仅以害虫免疫系统为靶标的新型绿色真菌杀虫剂的研制有重大指导意义，也为通过调控害虫的免疫系统来控制害虫奠定基础。

昆虫免疫真菌的侵染，是一个复杂的分子动态过程，体现在免疫基因表达策略。为阐明重大蔬菜害虫小菜蛾免疫虫生真菌的分子机制，本项目测定了虫生真菌侵染小菜蛾幼虫全转录组，结合在测定小菜蛾幼虫、蛹和成虫转录组及蛋白质组的基础上，筛选了小菜蛾免疫虫生真菌的免疫相关microRNA、mRNA和蛋白，构建了小菜蛾免疫虫生真菌的动态microRNA、mRNA和蛋白网络系统；对microRNA和mRNA的关联性进行了动态分析，预测了其靶标相关性，初步确定了免疫相关microRNA的靶标及其在免疫系统中的功能。具体结果如下：.1、.利用转录组学结合蛋白质组学筛选了调控小菜蛾免疫和发育相关基因，RNAi结合生化实验明确了PxSeripin7通过调控酚氧化酶PO的活力抵御虫生真菌的侵染，从而参与了小菜蛾的发育与免疫；.2、.蛋白质组学筛选了小菜蛾幼虫免疫虫生真菌Isaria fumosorosea侵染的关键蛋白GNBP3和PGRP-SA,RNAi明确GNBP3和PGRP-SA主要调控了抗菌肽的表达而抵御虫生真菌的侵染，并成功构建了沉默GNBP3和PGRP蛋白编码的重组dsRNA真菌和细菌；.3、.全转录组测定并筛选了小菜蛾幼虫免疫绿僵菌Metarhiziu manisopliae的microRNA, mRNA,lncRNA和cirular RNA,为小菜蛾免疫和绿僵菌的互作提供了核酸调控网络；.4、.RNAi结合扫描电镜和透射电镜明确了小菜蛾抗菌肽Moricin主要破坏真菌的细胞膜而杀死真菌；.5、.筛选并确定了miR-8545-x靶向serine proteinase9、trypsin3；miR-199-x与miR-122-x靶向serpin3； miR-283-3p靶向trypsin3； miR-276靶向βGRP从而调控酚氧化酶级联反应和抗菌肽来免疫绿僵菌及分泌的Destruxin。.通过本项目研究发表12篇SCI文章，申请专利5个,获得授权2个，培养硕士研究生11名，博士3名，博士后2名。