Cloning And Sequence Analysis Of The Metabolism-related Liver Genes In Orange-spotted Grouper (Epinephelus Coioides)

The liver, the biggest functional gland in organisms, participates in in vivo digestion, metabolism, excretion, disintoxicating and immunity processes and plays an important role in metabolism function. In order to study the genomic structure and function of orange-spotted grouper (Epinephelus coioides) amylase (AMY), trypsinogen (TRY), hepatic lipase (HL), liportein lipase (LPL), glutathione S-transferase alpha (GSTA) and glutathione S-transferase rho (GSTR) genes and the molecular mechanism of those genes in their molecular evolutional relationship in marine fish, the full-length cDNA sequences encoding AMY, TRY, HL, LPL, GSTA and GSTR were obtained from the liver of orange-spotted grouper(Epinephelus coioides) by RT-PCR and RACE methods. The obtained AMY was 1657 bp in length, encoding 512 amino acids. The deduced amino acid of amylase contains eight a-helical and eightβ-sheet structures, and the identities of amylase amino acid among fish, mammals and bird are 70.1-91.8%, which suggests that amylase is relatively more conserved. The cDNA of TRY was 911 bp, encoding 242 amino acids. The deduced amino acid sequence of trypsinogen has a catalytic triad and sequence analysis reveals that the identities of trypsinogen amino acid are 59.9-89.7% between fish and mammals. The sequence analysis showed that the complete cDNA sequence of HL of orange-spotted grouper was 2261 bp in length, encoding 494 amino acids. And the complete cDNA sequence of LPL was 2191 bp in length, encoding 515 amino acids. This research provides reliable evidences about the identity of amino acid sequence of HL and LPL and is useful for investigating physiology mechanism in relation to lipid metabolism. Sequence analysis reveals that, the identities of HL and LPL amino acid sequence among orange-spotted grouper, human, mouse and chicken, the identities of HL amino acid among orange-spotted grouper and mammals are 63.7-66.9% and the identities of LPL are 54.9-55.7%. The identities of HL and LPL reveal the higher similarity among teleosts later in the evolution (HL 61.9-73.1%, LPL 68.0-80.0%). Phylogenetic analyses revealed that, the deduced amino acid sequence of the orange-spotted grouper was more similar to largemouth bass and red sea bream than Chinese Sturgeon. The complete GST cDNA sequence of GSTA was 1008 bp in length, encoding 223 amino acids. And the complete cDNA sequence of GSTR was 1002 bp in length, encoding 225 amino acids. The sequence analysis showed that the deduced amino acid sequence of the GSTA was more similar to common carp and zebrafish than rat, mouse and human. Phylogenetic analyses revealed that, the deduced amino acid sequence of the orange-spotted grouper was higher similar to the GSTA of other fishes, such as common carp and zebrafish. The GSTR exists only in fish, but not in any other aquatic animals or mammals (e.g. rat, mouse and human). The research of digestion genes will help to further study the regulation of those genes, and develop dietary additives to stimulate their expression levels. The research of detoxification genes will help to directed selection high performance detoxication fish train and the further research and development of the new technology to enhance the marine fish edible safety.