MAVS在多倍体鱼天然免疫中的功能研究

The allotetraploid fish population was established first time in the world at Hunan Normal University, which is fertile for both sexes and owns high hereditary stability. A unique diploidy/triploidy/tetraploidy study platform is formed basing on the allotetraploid fish, diploid red crucian carp and their triploid hybrid-Xiangyun crucian carp. The antiviral immunity of this system is unknown. It is reported that MAVS (mitochondrial antiviral signaling protein) plays a major role in induction of the fish innate immune response against viruses. In this proposal, we plan to study the functional mechanism of MAVS in the antiviral immunity of the diploidy/triploidy/tetraploidy system, basing on the fibroblast cell lines to be established from the caudal fin of the fish with different ploidy. The research design includes: ① Establish and characterize the fibroblast cell lines from the diploidy/triploidy/tetraploidy; ② Molecular cloning and expression of the MAVS of the diploidy/triploidy/tetraploidy; ③Characterize the cellular distribution and expression of the MAVS of the diploidy/triploidy/tetraploidy; ④Characterize the antiviral function of MAVS in the diploidy/triploidy/tetraploidy. This study will elucidate the mechanism of MAVS in the antiviral immunity in the diploidy/triploidy/tetraploidy, which will built a solid foundation for the further study on the innate immunity of this polyploidy working system.

世界首例两性可育的异源四倍体鲫鲤群体在湖南师范大学被成功建立。异源四倍体鲫鲤、其二倍体父/母本和其三倍体杂交后代组成了一个亲缘关系明晰的二/三/四倍体鱼研发体系。目前有关该多倍体鱼体系的天然免疫抗病机制尚不清楚。最近的研究表明线粒体抗病毒信号蛋白（MAVS）在鱼类天然免疫抗病毒反应中起着重要的作用。本研究提出建立二/三/四倍体鱼体外培养成纤维细胞系，并籍此研究MAVS在多倍体鱼天然免疫系统中的功能的设想。本项目拟围绕该设想开展如下研究：① 建立和鉴定二/三/四倍体鱼尾鳍成纤维细胞系；② 克隆和表达二/三/四倍体鱼MAVS基因；③ 分析和鉴定MAVS在二/三/四倍体鱼成纤维细胞中的表达和定位；④ 研究MAVS在二/三/四倍体鱼成纤维细胞中抗病毒的功能。本项目将揭示MAVS在二/三/四倍体鱼天然免疫抗病毒反应中的功能机制，为将来系统研究二/三/四倍体鱼的天然免疫机制打下坚实的基础。

研究背景及研究内容.硬骨鱼类主要依靠天然免疫来抵抗病原微生物的入侵。世界首例两性可育的异源四倍体鲫鲤群体在湖南师范大学被成功建立。异源四倍体鲫鲤、其二倍体父/母本和其三倍体杂交后代组成了一个亲缘关系明晰的二/三/四倍体鱼研发体系。青鱼是我国重要的经济养殖鱼类。有关多倍体鱼体系及青鱼的天然免疫抗病机制尚不清楚。本研究建立了二/三/四倍体鱼及青鱼体外培养成纤维细胞系，并籍此对上述鱼类的RIG-I/MAVS/IFN信号通道在宿主天然免疫系统中的功能展开了系列研究。. .重要进展和关键数据：.1..建立了二倍体红鲫、三倍体湘云鲫、异源四倍体鲫鲤及青鱼尾鳍成纤维细胞系，目前这些细胞系均已培养至100代以上；对这些成纤维细胞系的染色体数目、倍增时间、DNA含量等进行了鉴定。.2..草鱼呼肠孤病毒（GCRV）及鲤春病毒血症病毒（SVCV）均能在上述细胞系中进行裂解复制，表明该四种细胞系可以成为研究宿主天然免疫抗病毒研究的极好模式。采用SVCV感染二/三/四倍体鱼成纤维细胞，采用GCRV感染青鱼尾鳍成纤维细胞；然后对上述细胞攻毒前后的转录组进行了测序和系统研究。.3..分别从二/三/四倍体鱼成纤维细胞中及青鱼尾鳍成纤维细胞中克隆了二/三/四倍体鱼及青鱼MAVS基因，对二/三/四倍体鱼MAVS及青鱼MAVS在宿主天然免疫抗病毒（诸如SVCV及GCRV）展开了系统研究。研究结果表明：二/三/四倍体鱼MAVS及青鱼MAVS均作用于宿主线粒体上；能够通过下游信号通道激活干扰素表达，从而引发宿主抗病毒天然免疫反应。.4..克隆了几种二/三/四倍体鱼干扰素基因，鉴定了其在宿主天然免疫中的抗病毒功能。.5..克隆了青鱼RIG-I、LGP2、STING、TRAF2/3/6、IKKe、TBK1、IRF3/7、Viperin、Mx1、IFN等基因；对它们在宿主天然免疫抗病毒反应中的功能进行了鉴定。为系统阐明青鱼天然免疫系统RIG-I/MAVS/IFN信号通道的抗病毒机制奠定了坚实的基础。