赤眼鳟与草鱼MDA5基因结构差异及其功能影响

The Ctenopharyngodon idellus is susceptible to death due to infection of a double-stranded RNA grass carp reovirus (GCRV), whilst the Squaliobarbus curriculus shows strong resistance to GCRV. The previous research found that the cytoplasmic pattern recognition receptor melanoma differentiation-associated gene 5 (MDA5) of S. curriculus and C. idellus participated in anti-GCRV immunity. The sequence difference is concentrated in C-terminal domain (plays a role in viral recognition) of MDA5 between two fish species. There is a double-stranded RNA (dsRNA) binding site in the C-terminal domain of MDA5 in S. curriculus, whereas C. idellus lacks the corresponding site. Based on the aforementioned results, this project is intended to further investigate: (1) the associations of difference in C-terminal domain of MDA5 with GCRV resistance; (2) the effects of difference in C-terminal domain and its dsRNA binding site on GCRV resistance; by applying the over-expression, RNA interference and site-directed mutagenesis techniques. This study aims to figure out the anti-GCRV immune function of C-terminal domain of MDA5, and to analysis and evaluate the functions of dsRNA binding site in antiviral immune response, and to provide molecular resources and new thoughts for selective breeding of C. idellus with strong viral resistance.

草鱼常因感染具有双链RNA基因组的草鱼呼肠孤病毒（grass carp reovirus, GCRV）致死，而赤眼鳟对GCRV具强抗性。前期研究发现赤眼鳟与草鱼胞质模式识别受体黑色素瘤分化相关基因5（melanoma differentiation-associated gene 5, MDA5）参与抗GCRV免疫。两种鱼MDA5序列差异集中于具病毒识别功能的C末端结构域；赤眼鳟C末端结构域含双链RNA结合位点，而草鱼缺乏该功能位点。在此基础上，本项目拟采用过表达、RNA干扰和点突变方法进一步深入研究:（1）MDA5基因C末端结构差异与GCRV抗性的关联；（2）C末端结构域及其双链RNA结合位点差异对GCRV抗性功能的影响。本项目旨在明确MDA5基因C末端结构域的GCRV抗性功能，分析和评价双链RNA结合位点的抗病毒作用，为抗病草鱼选育提供分子资源及新思路。

草鱼是中国水产养殖业产量最大的鱼，每年因GCRV感染而发生的草鱼出血病导致约20%的养殖损失。同属雅罗鱼亚科的草鱼和赤眼鳟GCRV抗性差异明显，且识别GCRV的胞质关键模式识别受体MDA5存在C末端结构的特定差异。基于此，通过项目实施我们克隆获得了赤眼鳟MDA5 3597bp的全长cDNA序列，可编码985个氨基酸残基。赤眼鳟MDA5在肝脏中蛋白水平最高，而在脾脏中最低；感染GCRV后MDA5蛋白水平呈现下降趋势。GCRV感染后赤眼鳟全部表现为抗性，草鱼则分为抗性及易感两类。MDA5 C末端序列差异主要体现在种间差异，即赤眼鳟MDA5有双链RNA结合功能位点,而草鱼缺乏该位点；草鱼MDA5 C末端无抗性关联位点。赤眼鳟MDA5 C末端蛋白能结合GCRV109核酸片段10（151-500 bp），且随蛋白量增加而结合能力增强，但草鱼MDA5 C末端蛋白未见结合。草鱼和赤眼鳟MDA5 C末端具有促进GCRV复制的功能，且草鱼MDA5 C末端能力更强；而赤眼鳟MDA5 C末端中dsRNA位点3的存在是促进病毒复制的关键因素。项目明确了草鱼和赤眼鳟MDA5结构差异对GCRV胞内扩繁的直接影响，发现了两种鱼MDA5 C末端结合GCRV核酸状态的差异，鉴定了赤眼鳟dsRNA结合功能区中影响GCRV扩繁的关键位点。MDA5 C末端主要是识别结合病毒核酸，然后通过CARD结构域诱导下游病毒感染免疫；我们的研究结果提示赤眼鳟和草鱼MDA5的C末端可能在GCRV大量扩繁期被利用来促进病毒复制。后续研究可深入解析MDA5与GCRV扩繁不同阶段的量效关系，并明确其功能属性的内在规律。