Molecular Characterization And Expression Responses Of TRAF6 And TAK1 To Immune Stimulations In Japanese Flounder(Paralichthys Olivaceus)

Japanese flounder (Paralichthys olivaceus) is benthic fish, which is one of the most economic important marine fish of china. In recent years, with rapidly developing of high density of intensive aquaculture, outbreaks of diseases have influenced the Japanese flounder aquaculture industry and caused heavy economic losses. Therefore, it is important to study the immune mechanism of Japanese flounder and explore the new prevention strategies and treatment approaches.Toll-like receptors (TLRs) play an important role in the innate system by recognizing conserved pathogen-associated molecular patterns (PAMPs). Upon recognizing PAMPs, TLRs can trigger the signaling pathways that activate immune cells in response to pathogen infection. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are two important adaptor molecules in Toll-like receptor signaling pathways. In the present study, we cloned the cDNA sequences of TRAF6 and TAK1 from Japanese flounder and detected the constitutive and induced expressions of these two genes. Additionally, the expressions of PoTRAF6 and PoTAK1 at different developmental stages were examined to find out the possible function of PoTRAF6 and PoTAK1 during flat fish development.The full-length cDNA of PoTRAF6 was 1953 bp, including a 5’-untranslated region (UTR) of 159 bp, a 3’-UTR of 81 bp, and an open reading frame (ORF) of 1713 bp. The ORF encoded a putative protein of 570 amino acids.The deduced amino acid sequence of PoTRAF6 protein is remarkably conserved. It shared significant homology with TRAF6s from other fish species. Among them, PoTRAF6 was most similar to Epinephelus tauvina (91%). The phylogenetic trees showed that PoTRAF6 shared the closest evolutionary relationship with TRAF6 of Cynoglossus semilaevis PoTRAF6 possessed a characteristic TRAF6 domain architecture composed of one N-terminal RING domain, two zinc fingers and one coiled-coil region followed by one conserved C-terminal meprin and TRAF homology (MATH) domain, which showed a high similarity to that of TRAF6 in other species. The conserved motifs of TRAF6 provide the probability of the similar functions with other mammal TRAF6s. The promoter region of PoTRAF6 was also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of TRAF6 were examined in developmental stages, different tissues and challenged samples. PoTRAF6 was expressed in all tissues and the high expressions of the gene were in the intestine and heart. The high levels of TRAF6 in intestine was consistent with the essential role of the two genes in TLR/IL-1R signaling pathway, which is pivotal in both innate and adaptive immune responses. PoTRAF6 expressed throughout the developmental stages. The expression of maternal mRNA throughout development suggested the potential role of TRAF6 in early immunity defense and developmental regulation of Japanese flounder. In challenged embryonic cells with LPS, CpG ODN or poly I:C, the expressions of PoTRAF6 were both up-regulated significantly. Those suggested that TRAF6 may play a crucial role in immune responses and might be involved in the process of development in Japanese flounder.The full-length PoTAK1 cDNA sequence was 2086 bp with a 5’-UTR of 232 bp, an ORF of 1728 bp and a 3’-UTR of 128 bp. The ORP encoded a protein sequence of 575 amino acid residues. Compared with other known TAK1s, the deduced amino acids sequence of PoTAK1 showed high similarity to that of Epinephelus coioides (97%). The phylogenetic trees showed that PoTAK1 shared the closest evolutionary relationship with TAK1of Cynoglossus semilaevis. Structure analysis of PoTAK1 indicated that it contained a protein kinases ATP-binding region signature, Serine/Threonine protein kinases active-site signature. Furthermore, a coiled-coil region was found at its C-terminus. The highly conserved domains indicated TAK1s have a similar function. The promoter region of PoTAK1 was also analyzed to predict several potential transcription factor-binding sites. In addition, the expression patterns of TAK1 were examined in developmental stages, different tissues and challenged samples. PoTAK1 was expressed in all tissues and the high expressions of the gene were in the intestine and heart. The high levels of TAK1 in intestine was consistent with the essential role of the two genes in TLR/IL-1R signaling pathway, which is pivotal in both innate and adaptive immune responses. PoTAK1 expressed throughout the developmental stages. The expression of maternal mRNA throughout development suggested the potential role of TAK1 in early immunity defense and developmental regulation of Japanese flounder. In challenged embryonic cells with LPS, CpG ODN or poly I:C, the expressions of PoTAK1 were both up-regulated significantly. Those suggested that TAK1 may play a crucial role in immune responses and might be involved in the process of development in Japanese flounder.