MOLECULAR CLONING AND CHARACTERIZATION OF TRYPSINOGEN GENE IN GOLDFISH (CARASSIUS AURATUS) AND ITS EXPRESSION PROFILES IN RESPONSE TO CADMIUM AND OXIDATIVE STRESS

Trypsin is a serine protease that plays a major role in protein digestion. It is synthesized and secreted by pancreas as a prepro enzyme, trypsinogen. In the present study, a full-length cDNA of goldfish trypsinogen (gfTryp) was successfully cloned from the hepatopancreas by rapid amplification of cDNA ends technique. The obtained gfTryp cDNA was 864 bp long with a 21 bp 5′-untranslated region (UTR), a 114 bp 3′-UTR containing the consensus polyadenylation signal AATAAA, and a 729 bp open reading frame encoding a protein of 242 amino acid residues. Sequence alignment showed that the gfTryp possessed all the characteristic features of trypsin family, suggesting its conserved function in protein digestion. Its mRNA expression was observed in all tissues examined, and the relatively higher levels were detected in hepatopancreas, intestine and fat. Hepatopancreatic gfTryp mRNA level decreased significantly after fasting for one week. Further periprandial expression analysis showed that gfTryp mRNA expression level in hepatopancreas dramatically up-regulated after meal. Hepatopancreatic gfTryp mRNA expression level increased significantly with 0.5 and 5 μg/mL cadmium (3.2 and 4.7 fold, respectively), and decreased with cadmium concentration up to 10 μg/mL. Strikingly, H₂O₂ exposure significantly decreased gfTryp mRNA expression in hepatopancreas at 3h, 6h, 12h and 24h. Our study provides the evidence that nutritional status, cadmium and oxidative stress can regulate the trypsinogen expression at the transcript level, and sheds new light on the physiological expression of trypsinogen in fish.