Molecular cloning and functional analysis of tumor necrosis factor receptor-associated factor 6 (TRAF6) inCrossastrea gigas

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Abstract

Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been demonstrated to be a key signaling molecule involved in adaptive and innate immunity. In this study, we obtained the full length CgTRAF6 cDNA and analyzed the characteristics of the ORF and the peptide sequence in Crassostrea gigas. The deduced protein sequence of CgTRAF6 includes a conserved C-terminal TRAF domain following the RING and the zinc finger domain. The TRAF domain is composed of coiled-coil TRAF-N and MATH (meprin and TRAF-C homology) subdomains. Furthermore, phylogenetic analysis revealed that CgTRAF6 is clustered together with other members TRAF6 family and is placed in a sub-cluster singly which had a close relationship with Drosophila melanogaster. Expression analysis of CgTRAF6 indicated its constitutive expression in all tissues including mantle, adductor muscle, digestive tract, gonads, heart, gill, and hemocyte. Immune challenge with Vibrio alginolyticus and poly I:C resulted in significant up-regulation of CgTRAF6 expression. Dual-luciferase reporter assays showed that CgTRAF6 could activate both pNF-κB-Luc and pISRE-Luc expression, suggesting CgTRAF6 is potentially involved in NF-κB and the interferon signaling pathway. Furthermore, RNAi mediated knockdown of CgTRAF6 resulted in the down-regulation of several putative anti-viral signaling (IRF) and effector (PKR & Viperin) molecules coding genes, 7 days post-injection. These results collectively indicate that CgTRAF6 is a member of TRAF6 sub-family and is potentially involved in immune defense system against invading bacteria and viruses in Crassostrea gigas.

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