草鱼TAK1信号传导及其抵御病原感染的功能研究

Transforming growth factor-β-activated kinase 1 (TAK1) is a key signal molecule involved many innate immune signaling pathways, including TLR, IL-1, TNF, BCR, and TCR pathways. TAK1 plays a crucial role in host immune response via the stringent control of NF-κB and AP-1 signal. Significant progress has been made in clarifying the role of TAK1 in mammals, but the function of TAK1 is still poorly understood in fish. Our early works have indicated that TAK1 may play a crucial role in the immune response against pathogen infection. In this study, we will do further research on the signal transduction and immune defense function of TAK1 in grass carp (Ctenopharyngodon idella). The intracellular localization of full-length TAK1 and its domains is detected by Laser Confocal Microscope. The over-expression vectors are constructed, including full-length TAK1, a series of domains, and some site mutants, and then are transfected into CIK cells. Activation of NF-kB and AP-1 induced by each recombined plasmid is measured using the luciferase reporter assay. The interaction of TAK1, TAB1, and TAB2 is analyzed by co-immunoprecipitation and luciferase reporter assay. These results will preliminarily elucidate the signal transduction mechanism of TAK1. After grass carp reovirus (GCRV) infection and viral/ bacterial PAMPs stimulation in CIK cells transfected with the expression plasmids, including full-length TAK1, some domains, site mutants, and shRNA, the antiviral and antibacterial roles are compared. Besides, the mRNA expression of several genes in TAK1 signaling pathway are detected in the immune system organs and local infection sites after infection with GCRV and Flavobacterium columnare. These results collectively will clarify the functional characterizations of TAK1 in host defense against pathogen infection. Above all, the results of this project will provides a scientific basis for further recognition of the immune signaling pathways in fish, the study of disease resistance mechanisms, and the development of management strategies for disease control.

转化生长因子β活化激酶1（TAK1）是调控TLR、IL-1、TNF等信号通路的关键节点蛋白，在先天性免疫防御中起重要作用。在哺乳动物中TAK1的功能特征已基本明晰，但是目前关于鱼类TAK1的研究仍非常匮乏。本研究以草鱼TAK1为对象，在前期研究基础上，确定TAK1亚细胞定位及决定其细胞定位的结构域，揭示TAK1全长、结构域及关键位点对NF-κB、AP-1活性的影响，并分析TAK1和TAB1、TAB2的蛋白相互作用，进而初步阐明TAK1的信号传导机制。通过草鱼呼肠孤病毒（GCRV）和病原相关分子模式（PAMPs）感染/刺激各类转染细胞，在细胞水平上阐明TAK1全长及结构域、关键位点的抗病毒和抗菌作用；同时检测GCRV和柱状黄杆菌感染草鱼后TAK1信号通路基因的表达，在个体水平上揭示TAK1抵御病原感染的功能。此研究结果为进一步认识鱼类免疫信号通路，研究抵御病原感染的先天性免疫机制奠定基础。

转化生长因子β活化激酶1（TAK1）是调控TLR、IL-1、TNF等信号通路的关键节点蛋白，在先天性免疫防御中起重要作用，但是目前关于鱼类TAK1的研究仍非常匮乏，基于此，本项目进行了草鱼TAK1信号传导及抵御病原感染的功能研究。草鱼TAK1及两种结合蛋白（TAB1和TAB2）都定位在细胞质中。过表达TAK1和TAB1都能显著激活NF-κB活性，存在一定的转染剂量依赖性，且激活能力具有平台期，而过表达TAB2对其活性无显著影响。较单一质粒转染，共转染TAK1和TAB1更能够显著增强NF-κB活性，而共转染TAK1和TAB2对NF-κB活性增强效果不明显。定量PCR检测嗜水气单胞菌感染后草鱼TAK1和TLRs、关键信号传导分子及免疫因子在不同组织不同时间点的基因表达情况，发现TAK1相关信号通路基因表达量受病原菌感染调节，主要表现为上调。比较了草鱼和斜带石斑鱼的信号通路基因IRAK1的信号传导功能，发现二者的过表达都能显著激活NF-κB活性，且分别在抵御多子小瓜虫和刺激隐核虫的感染中起到重要作用。进行了多子小瓜虫感染后在不同时间点草鱼鳃和脾的动态转录组学分析，鉴定了感染组和对照组的差异基因，并进行了GO分类及通路分析，筛选得到起关键作用的免疫相关信号通路和基因。同时，进行了多子小瓜虫感染后草鱼鳃的蛋白质组学（iTRAQ）分析，通过差异蛋白分析，筛选获得重要的信号通路和关键蛋白。通过以上研究，初步阐明了草鱼TAK1的信号传导机制，揭示了TAK1相关信号通路在抵御病原感染中的作用，具有重要学术价值。