Full-length cDNA cloning of IgM heavy chain of cobiaRachycentron canadiumand quantitative analysis in different tissues

Abstract

Full-length cDNA of IgM haevy chain of cobia (Rachycentron canadium Linnaeus) was ampli-fied by the techniques of Rapid Amplification of cDNA ends (RACE) and PCR, and expression of target gene was analyzed by the method of real time RT-PCR in different tissues. The full-length cDNA of μ of cobia is 1 933 bp, with a 5?terminus untranslated region (UTR) of 26 bp and a 3?terminus UTR of 164 bp, carried an open reading frame (ORF) of 1 743 bp, encoding 580 amino acids with estimated molecular weight of 64.83 ku and the theoretical isoelectric point of 6.6. The similarity of the deduced Igμ amino acid sequences of cobia was less than 30%, when compared with those of other animals. Moreover, similarities of the conservative CH4 region of cobia,to other fish were 64%-71%. But, to other living things, similarity was only 31%-33%, having a strong specificity. Clustal X analysis indicated that there existed conserved amino acid sites (cysteines, tryptophans) and two E-box motifs GKGLEW at framework region two and YYCAR at framework region three, respectively in the IgM heavy chain of cobia. Ig μ gene was ex-pressed in all the tissues but kidney in healthy cobia and the expression with the highest quantity was pre-sent in intestine by real time RT-PCR. The target gene was respectively expressed in all tissues of cobia after stimulation by intraperitoneal injection with Vibrio carchariae for 48 h, 96 h and 192 h. The expres-sion of Igμ gene was significantly increased in heart, gill and intestine after 48 h. Increased expression of gene in spleen, stomach, brain was after 0 h and in liver, head kidney and kidney was after 192 h. Conclu-sion could be drawn that mucosal immune defended against foreign pathogens as the first barrier, followed by the liver, spleen, kidney, head kidney immune response, which indicated that immune system plays an important role in defense against foreign pathogens for a long time.