Molecular Cloning And Functional Characterization Of Microcystin Detoxification-related Liver Genes In Freshwater Fishes

Cyanotoxins pose a world-wide health threat to animals and humans due to their increasing presence in both drinking and recreational waters. Microcystin detoxification-related liver genes, such as soluble glutathione S-transferase (sGST) and uncoupling protein 2 (UCP2) have been supposed to play an important role in microcystin detoxification in fish body. In this report, molecular cloning and structure characterization of liver GST and UCP2 gene, from major Chinese freshwater fishes with various food habit, including silver carp (Hypophthalmichthys molitrix) (phytoplanktivorous fish), bighead carp (Aristichthys nobilis) (zooplanktivorous fish), grass carp (Ctenopharyngodon idellus) (herbivorous fish), crucian carp (Carassius auratu) (benthos-eating fish), mud carp (Cirrhinus molitorella) (detritus-eating fish), and tilapia (Oreochromis nilotica) (phytoplanktivorous fish), were carried out. In addition, liver GST and UCP2 gene expression levels among these freshwater fishes were also studied, which would shed a new light on the detoxification mechanism of microcystins in freshwater fishes.Two full-length cDNAs encoding GST were cloned and sequenced from the hepatopancreas of planktivorous silver carp and bighead carp. The silver carp and bighead carp GST cDNA were 920 bp and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from grass carp, crucian carp, mud carp and tilapia liver. All these GST could be classified as alpha-class GST on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using computer, and found to fit the classical two-domain GST structure. Using genome walker method, a 875 bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish, might be regulate by LPS, released from the toxic blue-green algae producing microcystins.