Molecular Cloning And Functional Characterization Of Microcystin Detoxification-related Liver Genes In A Phytoplanktivorous Fish, Nile Tilapia (Oreochromis Niloticus)

Liver genes related to phase I and phase II detoxification, as well as inhibition of reactive oxygen species (ROS) production, were cloned, and their response to microcystin-LR (MC-LR) and lipopolysaccharide (LPS) exposure via intraperitoneal injection, was determined in a phytoplanktivorous fish, Nile tilapia (Oreochromis niloticus).The cloned full-length cDNA of tilapia soluble glutathione S-transferase (sGST) was classified as alpha-class GST on the basis of its amino acid sequence identity with other species. The tilapia sGST clone was 861 bp in length, and contained a 25 bp 5'-UTR, a 167 bp 3'-UTR and an open reading frame of 669 bp, encoding a polypeptide of 222 amino acids. Using genome walker method, a 366 bp 5'-flanking sequence of tilapia sGST gene was further obtained, and the possible regulatory elements were identified. Partial cDNA sequences of glutathione peroxidase (GPX) and uncoupling protein 2 (UCP2) were also obtained by PCR using degenerate primers from tilapia liver.To study the transcriptional response of liver genes to microcystin treatment, tilapia were respectively exposed to a single 50 μg kg^-1 body weight (bwt) dose of pure MC-LR, a single 2 mg kg^-1 bwt dose of LPS and a co-exposure MC-LR and LPS (50 μg kg^-1 bwt + 2 mg kg^-1 bwt), and were then sacrificed at 24 h post-exposure. Using beta-actin as external control, a significant increase (about 80%) in sGST mRNA expression was found in response to the MC-LR exposure after 24 h (P < 0.05), indicating the importance of sGST in microcystin detoxification. A slight decrease of sGST mRNA expression was observed in the liver of tilapia, exposed to LPS and MC-LR+LPS. It seems that the LPS response element (LPSRE), identified in the promoter region of tilapia sGST gene, might be functional at a rather low level. Although not significant, the mRNA expression level of...