Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao, 266071, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China; College of Fisheries and Life, Shanghai Ocean University, Shanghai, China.
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao, 266071, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China.
Fish Shellfish Immunol. 2020 Jan;96:13-25. doi: 10.1016/j.fsi.2019.11.029. Epub 2019 Nov 21.
Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) play crucial roles as signaling mediators for the TNF receptor (TNFR) superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAFs collectively play important roles in multiple biological processes and organismal immunity. However, systematic identification of the TRAF gene family in teleost fish has not yet been reported, and there is little available information about its roles in innate immunity in Chinese tongue sole (Cynoglossus semilaevis), an aquaculture fish of high economic value. In the present study, we identified and characterized seven TRAF genes, namely, CsTRAF2a, CsTRAF2b, CsTRAF3, CsTRAF4, CsTRAF5, CsTRAF6 and CsTRAF7, in Chinese tongue sole, and the complete ORFs of the CsTRAFs were cloned. Sequence analysis revealed various genomic structures of the CsTRAFs and showed that they contain typical conserved domains compared with mammalian TRAFs. Phylogenetic analysis indicated the evolutionary relationships of TRAF family members in teleost fish and revealed an absence of TRAF1 in most species and TRAF5 in some species of teleosts. Analysis of the gene structures and motifs showed the diversity and distribution of exon-intron structures and conserved motifs in Chinese tongue sole and several other teleost species. Real-time quantitative PCR was used to investigate the expression patterns of CsTRAF genes in tissues of healthy fish and in the gills, livers and spleens of fish after bacterial infection with Vibrio harveyi. The results indicate that only CsTRAF2a is relatively highly expressed in the brain and that the other CsTRAFs are highly expressed in immune-related tissues and may participate in the immune response after infection with pathogenic bacteria. Functional analysis of CsTRAF3, CsTRAF4 and CsTRAF6 revealed that only CsTRAF6 could strongly activate the NF-кB pathway after overexpression of CsTRAF3, CsTRAF4 and CsTRAF6 in HEK-293T cells. This systematic analysis provided valuable information about the diverse roles of TRAFs in the innate immune response to pathogenic bacterial infection in teleost fish and will contribute to the functional characterization of CsTRAF genes in further research.
肿瘤坏死因子（TNF）受体相关因子（TRAFs）作为 TNF 受体（TNFR）超家族和白细胞介素-1 受体/Toll 样受体（IL-1R/TLR）超家族的信号转导介质，发挥着至关重要的作用。TRAFs 共同在多种生物过程和机体免疫中发挥重要作用。然而，鱼类 TRAF 基因家族的系统鉴定尚未见报道，关于其在中国舌鳎（Cynoglossus semilaevis）先天免疫中的作用的信息也很少，中国舌鳎是一种具有高经济价值的养殖鱼类。在本研究中，我们在舌鳎中鉴定并表征了七个 TRAF 基因，即 CsTRAF2a、CsTRAF2b、CsTRAF3、CsTRAF4、CsTRAF5、CsTRAF6 和 CsTRAF7，并克隆了 CsTRAFs 的完整 ORF。序列分析显示 CsTRAFs 的各种基因组结构，并表明它们与哺乳动物 TRAFs 相比含有典型的保守结构域。系统进化分析表明了鱼类 TRAF 家族成员的进化关系，并显示出大多数鱼类中缺乏 TRAF1 和一些鱼类中缺乏 TRAF5。基因结构和基序分析显示了中国舌鳎和其他几种鱼类中exon-intron 结构和保守基序的多样性和分布。实时定量 PCR 用于检测 CsTRAF 基因在健康鱼类组织以及感染哈维弧菌后鱼类鳃、肝和脾中的表达模式。结果表明，只有 CsTRAF2a 在脑中相对高表达，而其他 CsTRAFs 在免疫相关组织中高表达，可能参与感染病原菌后的免疫反应。CsTRAF3、CsTRAF4 和 CsTRAF6 的功能分析表明，只有 CsTRAF6 能够在 HEK-293T 细胞中转染 CsTRAF3、CsTRAF4 和 CsTRAF6 后强烈激活 NF-кB 通路。这项系统分析为 TRAFs 在鱼类对病原菌感染的先天免疫反应中的多种作用提供了有价值的信息，并将有助于进一步研究中 CsTRAF 基因的功能特征。
