Studies On Molecular Cloning And Tissue Expression Of Glutathione Peroxide Of Silver Carp Hypophthalmichys Molitrix Bighead Carp Aristichthys Nobilis And Grass Carp Ctenopharyngodon Idella

Glutathione peroxidase (GPX) is a generic name for a family of multiple isozymes. In general, the members in the family can be divided into two sorts. One sort of the isozymes have selenium-dependent glutathione peroxidase activity and contain selenocysteine (SeC) encoded by a TGA codon, while the other sort have no selenocysteine. In this study, glutathione peroxidase (GPX) cDNAs were cloned from the liver of phytoplanktivorous freshwater fish, including silver carp Hypophthalmichthys molitrix and bighead carp Aristichthys nobilis, and herbivorious fish, grass carp Ctenopharyngodon idella using RT-PCR. Sequence analysis revealed that the cloned GPX cDNA fragments were all 263bp in length. The 3'-cDNA sequences of bighead carp and grass carp GPX were further obtained by using the method of 3'-rapid amplification of cDNA ends (RACE). The incorporate GPX cDNA fragment of bighead carp was 653bp in length, and that of grass carp was 653bp in length. Then the full-length GPX cDNA sequence of silver carp was further obtained by using the method of 3'-and 5'-rapid amplification of cDNA ends (RACE). With the method of the Genome Walker, a sequence of 750bp of 5'-flanking region of silver carp GPX gene was obtained. The silver carp GPX cDNA were 892 bp in length, containing an open reading frame (ORF) of 576 bp (encoding a polypeptide of 191 amino acids), flanked by 25 bp 5'-untranslated region (5'UTR) and 291 bp 3'-untranslated region (3'UTR). The theoretical molecular weight (M_W) of the putative peptide was estimated to be 21.59 kDa. The 40th amino acid corresponds to a selenocysteine encoded by a TGA codon. Search results of the BLAST protein database revealed that the deduced amino acid sequences of silver carp, bighead carp and grass carp GPX were highly (75-93ï¼…) homologous with those of other vertebrates (including zebrafish, rock bream, rainbow trout, human, mouse, rat, pig and cow). Phylogenetic analysis suggested that these three Chinese carp GPXs belong to GPX2. However, unlike mammalian GPX2, which has been shown to locate mainly in the gastrointestinal tract, the GPX2 gene expression of silver carp was found in the liver, adipose tissue, intestine, muscle and brain. Using beta-actin as control, the comparison liver GPX expression of the three freshwater fish species showed a close relationship with their food habit, and that is the expression in planktivorious fish (silver carp and bighead carp) is stronger than that in herbivorious fish (grass carp), suggesting that liver GPX might be important for fish to reduced lipid peroxides which are induced by microcystins.