Cloning, Promoter Analysis And Expression Of The Tumor Necrosis Factor Receptor-associated Factor6(TRAF6) In Japanese Scallop (Mizuhopecten Yessoensis)

The Japanese scallop (Mizuhopecten yessoensis) is an economically important marinemolluscs found along the northern part of the Korean Peninsula, the coasts of the northernislands of Japan, and Kuril islands and the Sakhalin. M. yessoensis was introduced to Chinafrom Japan in1982, since then, its cultivation in China has developed rapidly. In recent years,due to the degeneration in inbreeding, seawater pollution and high stocking density, anoutbreak large number of M. yessoensis deaths, which not only caused a huge economic loss,but also seriously affected the sustainable development of this aquaculture.Tumor necrosis factor receptor-associated factor6(TRAF6) is a key adaptor molecule forthe tumor necrosis factor superfamily and Toll-like/interleukin-1receptor superfamily. Itplays an important role in innate and adaptive immunity. The TRAF6of Japanese scallopMizuhopecten yessoensis (designated as MyTRAF6) was identified and characterized in thisstudy. The full-length cDNA of MyTRAF6was2407bp by3′RACE, which consisted of239-bp5′-terminal untranslated region,1974-bp open reading frame (ORF) encoding apolypeptide of657amino acids,194-bp of3′-terminal UTR followed by acanonicalpolyadenylation signal sequence AATAAA and a poly (A) tail. The predicted aminoacid sequence of MyTRAF6contained the characteristic motifs of TRAF proteins, including aZinc finger of RING-type, two Zinc fingers of TRAF-type, and a MATH (meprin and TRAFhomology) domain. It had an overall identity of43-96%with those of other TRAF6s, thehighest identity (96%) with Chlamys farreri TRAF6, and the least identity (43%) withMeleagris gallopavo TRAF6. Phylogenetic analysis classified MyTRAF6as a true TRAF6ortholog.A1462bp of promoter sequence was obtained by genome walking. Analysis of theputative promoter region by TRANSFAC and AliBata2.1revealed numerous possible bindingsites for transcription factors. Three putative SNPs of MyTRAF6were identified when the1462-bp DNA fragments amplified from the MyTRAF6promoter region of15individual M.yessoensis were aligned with each other. These SNPs were found in positions615(C/G),553(T/C) and419(G/A)。For the615C/G-and419G/A-specific alleles, no changes inthe TFBSs were detected. As for the553T/C-specific allele, one putative TFBS was presentin the T-specific allele, but was absent in the C-specific allele.Quantitative RT-PCR analysis revealed that MyTRAF6was expressed in all the adult M.yessoensis tissues and challenge by the bacteria Vibrillo anguillarum. The results reveal thatMyTRAF6was highly expressed in hemocytes of adult M.yessoensis. MyTRAF6transcript level in the hemocytes reached a maximum six hour after Vibrio anguilarum challenge. Theresults indicated that MyTRAF6may fulfill an important function during M. yessoensisbacterial infection. It could be a key effector molecule involved in the innate defense ofmolluscs.This study will serve as a step forward in increasing our understanding of the immunesystem of scallop, as well as the ultimate goal of finding a way to combat the influences ofdiseases that could threaten the farming of scallops.