MOLECULAR CLONING AND IN VIVO EXPRESSION ANALYSIS OFMICROCYSTIN DETOXIFICATION-RELATED GENES IN NILE TILAPIA (OREOCHROMIS NILOTICUS)

Soluble glutathione S-transferase (sGST) of freshwater fish is extremely important to microcystins (MCs) purification from fish body, by catalyzing the conjugation of GSH (Glutathione) with microcystins. Glutathione peroxidase (GPX) is essential to the detoxification of cyanotoxins by providing GSH for sGST. Because oxidation and reactive oxygen species (ROS) generation is necessarily involved in both the toxication and detoxification process of cyanotoxins in hepatocytes, uncoupling protein 2 (UCP2) also has an important role in inhibiting the excessive production of ROS to restrain hepatocytes apoptosis. In this study, RT-PCR using degenerated primers, yielded a sGST cDNA fragment of 399 bp, a GPX cDNA fragment of 280 bp and a UCP2 cDNA fragment of 776 bp from the liver of a phytoplanktivorous freshwater fish, nile tilapia (Oreochromis niloticus) , which consumed substantial amounts of toxic blue-green algae in the food. The sGST cDNA fragment was further completed by 5. and 3. RACE (Rapid amplification of cDNA ends) . The full length tilapia sGST cDNA was 861 bp in length, containing an ORF (Open reading frame) of 669 bp (encoding 222 amino acids) , flanked by 25 bp 5. UTR (Untranslated region) and 167 bp 3. UTR. The deduced amino acid sequence from this sGST cDNA fragment contains two conserved domains, N-terminal domain (glutathionebindind site) and C-terminal domain (substrate-binding site) . Homology of the sGST amino acid sequence is high (64. 3%) 78. 5%) with red sea bream (Pagrus major) , rock bream (Oplegnathus fasciatus) and zebrafish sGST, and is low (51. 8%) 55. 9%) with human, rat, cow, pig and chicken sGST. However, both the GPX and UCP2 amino acid sequences show a high conservation with GPX and UCP2 of both fish and mammals (69.6%) 85.9% for the GPX with rainbow trout, rock bream, zebrafish, human, rat, mouse, cow and pig GPX, and 71.8%) 93.8% for the UCP2 with red sea bream, zebrafish, common carp, European chub Leuciscus cephalus, grass carp, human, rat and mouseUCP2) . T ilapia juveniles (5) 8 g) were exposed to a sub-lethal dose (50Lg/kg bwt) of MC-LR by intraperitoneal injection. Using B-actin as external control, a significant increase (about 80%) in the liver sGST mRNA expression was found in response to the MC-LR exposure after 24h (p< 0105) , indicating the importance of GST in microcyst in detoxification. Although no significant changes were seen in the liver GPX and UCP2 mRNA expression, the expression level of both genes tended to increase after exposed toMC-LR. We suggested that sGST might be responsible for the strong tolerance of the phytoplanktivorous fish to microcystins, and hepatocyte proteins coping with oxidative stress (GPX and UCP2) , might also have some auxiliary effect.