假胚分泌的microRNA保护腰带长体茧蜂幼虫逃避寄主包囊反应的功能机制研究

内寄生蜂在与寄主协同演化的过程中，进化出了多种调控寄主的生理活动以实现成功寄生的策略。假胚是腰带长体茧蜂Macrocentrus cingulum产生的一种胚胎样合胞体。前期研究表明假胚能抑制寄主对蜂幼虫的包囊，本项目拟阐明假胚调控寄主针对蜂幼虫的免疫反应的功能机制。首先结合胚胎移植、胚胎体外培养及电镜负染等技术分析假胚对寄主免疫反应的抑制功能以及miRNAs的来源；然后利用miRNA的模拟物，从高通量测序结果中筛选出来自假胚且能抑制蜂幼虫的包囊的miRNAs，并利用双荧光报告系统验证它们的靶基因及结合位点；再结合抗体封闭、RNAi、CRISPR/cas9及重组蛋白功能等分析证明靶基因的功能。最终阐明假胚通过分泌miRNAs调控寄主体内的靶基因的表达，抑制寄主对蜂幼虫的包囊的分子机制。该研究将填补寄生蜂子代调控寄主的免疫反应的研究空白，为系统阐明寄生蜂调控寄主生理活动的机制奠定理论基础。

During the coevolution of endoparasitoids and their hosts, a variety of strategies have been developed in parasitoids to regulate the physiological activities of hosts to achieve successful parasitization. Pseudogerm is a kind of embryo-like syncytium produced by the endoparasitoid, Macrocentrus cingulum. Previous studies showed that pseudogerm can protect the larvae of M. cingulum from being encapsulated by host’s hemocytes. This study intends to elucidate the functional mechanisms of pseudogerm regulating the immune reactions of host. First, the immunosuppressive function of pseudogerm and the source of miRNAs are analyzed by embryo transfer, in vitro culture and negative staining in electron microscope. Then, miRNAs from pseudogerms inhibited the encapsulation of the larvae of M. cingulum are screened out in miRNAs from host hemolymph acquired by high-throughput sequencing by the injection of miRNA agomir; Then, the target genes and binding sites of miRNAs are identified by Dual Fluorescence Reporting System. Furthermore, the functions of miRNAs are analyzed by antibody blocking, RNAi, CRISPR/cas9 and the function of recombinant protein. Finally, the molecular mechanisms that how miRNAs secreted by pseudogerm regulate the expression of its target genes, and inhibit the encapsulation of host on wasp larvae are clarified. This study will fill in the research gaps in knowledge regarding the regulation of host immune response by parasitoids' offspring, and provides an important theoretical basis for the systematic elucidation of the mechanism that how parasitoids regulates the physiological activities of their host.