草鱼miR-200b靶向NLRC3的免疫调控机制

草鱼呼肠孤病毒（grass carp reovirus, GCRV）感染引起的草鱼出血病严重制约草鱼产业的健康发展；miRNA可靶向调控天然免疫分子及下游通路参与机体的免疫应答。申请人发现miR-200b可明显加速草鱼卵巢细胞系中GCRV引起的细胞病变，并搜索到NLRC3是miR-200b的预测靶基因。本项目拟聚焦miR-200b靶向NLRC3在GCRV免疫应答过程中调控IFN反应的分子机制，开展下列主要研究：1）结合全长转录组、双荧光素酶活性分析及qPCR等方法确证miR-200b与NLRC3的靶向关系； 2) 采用过表达/敲降和病毒感染后的细胞病理学、病毒繁殖动力学及分子免疫学等分析，研究草鱼miR-200b与NLRC3在GCRV免疫应答中的效应；3）利用co-IP等蛋白实验验证miR-200b通过NLRC3介导调控IFN反应的分子机制。为抗病草鱼的分子辅助育种奠定有效途径。

The hemorrhagic disease caused by grass carp reovirus (GCRV) seriously restricts the healthy development of the grass carp industry. miRNA could target innate immune molecules and regulate downstream pathways to participate in immune response. Previous study found over-expression of miR-200b can obviously accelerate the cytopathic effect (CPE) caused by GCRV infection in grass carp ovarian cell line, and NLRC3 is a potential target of miR-200b. This project intends to focus on the regulatory mechanism involving in miR-200b/NLRC3 regulating IFN response in immune response to GCRV, carry out the following research: 1) elucidate the direct targeting interaction relationship between miR-200b and NLRC3 using full-length transcriptome, dual luciferase activity analysis and qPCR confirmation; 2) confirm the effect of miR-200b and NLRC3 in GCRV immune response by analysis of cellular pathology, virus reproductive dynamics and molecular immunology; 3) uncover the molecular mechanism of miR-200b regulating IFN through targeting NLRC3 with co-IP and other protein experiments. This study will provide an effective way for molecular assisted breeding of highly resistant grass carp.