Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, Ministry of Agriculture, Shanghai, 201306, China.
Key Laboratory of Genetic Resources for Freshwater Aquaculture and Fisheries, Shanghai Ocean University, Ministry of Agriculture, Shanghai, 201306, China; Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China; Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai, 201306, China.
Fish Shellfish Immunol. 2018 Sep;80:335-347. doi: 10.1016/j.fsi.2018.06.035. Epub 2018 Jun 18.
Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.
肿瘤坏死因子受体相关因子 6（TRAF6）作为一种中枢细胞内信号接头分子，在脊椎动物和无脊椎动物中介导肿瘤坏死因子受体超家族和白细胞介素 1 受体/Toll 样受体家族。本研究从中国淡水蛏（Hyriopsis cumingii）克隆并鉴定了一种软体动物同源物 HcTRAF6。HcTRAF6 的完整开放阅读框包含一个 1965bp 的区域，编码一个 654 个氨基酸的预测蛋白，该蛋白包含保守的特征结构域，包括一个 RING 结构域、两个 TRAF 型锌指结构域、一个典型的卷曲螺旋和 MATH 结构域。系统发育分析表明，HcTRAF6 与中华圆田螺（Cyclina sinensis）的 CsTRAF6 在无脊椎动物软体动物聚类中紧密聚集。进一步的 qRT-PCR 分析表明，HcTRAF6mRNA 在贻贝组织中广泛分布，在鳃中表达量较高。在受到嗜水气单胞菌和脂多糖免疫刺激后，HcTRAF6 在鳃和血细胞中的转录明显被诱导。此外，在珍珠囊、鳃和血细胞中观察到 HcTRAF6 表达在套膜植入后有明显波动。这些发现证实了其在同种免疫反应中的作用。双荧光素酶报告基因检测表明，HcTRAF6 的过表达可以以剂量依赖的方式增强 NF-κB 报告基因的活性。进一步的 RNA 干扰表明，在 HcTRAF6 沉默贻贝中，抗菌肽在抗菌感染中的上调被强烈抑制，而 HcTRAF 的耗竭抑制了嗜水气单胞菌的消除。所有这些发现共同证明，HcTRAF6 在中华淡水蛏对抗入侵病原体的固有免疫机制和套膜植入后的同种免疫机制中发挥有效的调节作用。
