MOLECULAR ISOLATION AND CHARACTERIZATION OF THE TUMOR NECROSIS FACTOR RECEPTOR-ASSOCIATED FACTOR 6 (TRAF6) FROM Macrobrachium rosenbergii

Abstract

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein that can be participated in both tumor necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL1/TLR) superfamily. TRAF6 is involved in various biological processes such as development of T-cells, cell proliferation, apoptosis, as well as innate and adaptive immunity. In this study, a novel TRAF6 gene was identified and characterized from Macrobrachium rosenbergii, designated as MrTRAF6. The full-length cDNA of MrTRAF6 was 2,114 nucleotides long with an open reading frame (ORF) of 1,695 nucleotides that encoded a protein of 564 amino acid residues. The MrTRAF6 protein consisted of two RING type Zinc fingers, and a C-terminal meprin and TRAF homology (MATH) domain. The amino acid sequence of MrTRAF6 shared 46.1-87.6% identity with TRAF6s from other crustacean species with the highest identity to TRAF6 of Macrobrachium nipponense. Phylogenetic analysis indicated that MrTRAF6 showed a close relationship with TRAF6 proteins of invertebrates and formed a cluster with other crustacean TRAF6 proteins. Tissue distribution and expression analysis demonstrated that MrTRAF6 transcript was widely expressed in all tissues examined, with the highest expression level in the gills and the lowest in muscle tissue. After challenge with Aeromonas hydrophila, MrTRAF6 was significantly upregulated in the muscle, hemocyte, gill, and hepatopancreas tissues. To investigate the specific role of MrTRAF6 in immunity, RNA interference (RNAi) was performed. The results showed that silencing of MrTRAF6 by dsRNA could reduce the expression of crustin, and mannose-binding lectin (MBL) but no significant change was observed in anti-lipopolysaccharide factor 5 (ALF5) level. Furthermore, the silencing of MrTRAF6 led to a significant increase in cumulative mortality rate after exposure to A. hydrophila. These results suggest that MrTRAF6 plays a crucial role in the innate immune response of M. rosenbergii, specifically in terms of antibacterial activity.-