Molecular Cloning And Expression Of Antioxidant Enzyme Genes From Zhikong Scallop Chlamys Farreri

Zhikong scallop Chlamys farreri is a commercially important cultrued species in China. The scallop industry has grown rapidly over the last 20 years. Since the large-scale mortality of cultured scallop happened in 1994, the frequently outbreaks of diseases have resulted in declined production of farmed scallop. Understanding the immunity of scallop may contribute to develop strategies for management of diseases and for long-term sustainability of scallop culture.Antioxidant enzymes that can remove reactive oxygen species and reduce oxidative stress play important role in immune system. In present study, large scale EST technique, homology-base technique and RACE technique were used to clone superoxide dismutase, catalase and glutathione peroxidase genes in Zhikong scallop C. farreri and the structures of these genes were analysized. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with L. anguillarum, M. luteus and C. lipolytica respectively.The full-length cDNA of CfSOD was of 1022bp with an ORF of 459bp encoding 153 amino acids without a signal peptide. BLAST analysis revealed that the predicated amino acid sequence of CfSOD shared high identity with Cu/Zn-SOD in other organisms. There were two Cu/Zn-SOD signature sequences in CfSOD, and the amino acids required for the Cu (His-45, -47, -62 and -119) and Zn (His-62, -70, -79 and Asp-82) were conserved in CfSOD. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD was down-regulated and then recovered after L. anguillarum and M. luteus challenged, but was not induced by C. lipolytica challenge.The full-length cDNA of CfCAT was of 3146 bp with an ORF of 1521bp encoding 507 amino acids without a signal peptide. BLAST analysis revealed that the predicated amino acid sequence of CfCAT shared high identity with CAT in other organisms. There were one catalase proximal active site signature and one catalase proximal heme-ligand signature sequence in CfCAT, and the peroxisomal targeting signal AQL were conserved in CfCAT. Higher-level mRNA expression of CfCAT was detected in the tissues of gonad and gill filaments. The expression of CfCAT was induced in the first 4 hours and reached the highest point (about 6.8 fold higher than blank group) in 4 hour point then recovered after L. anguillarum challenged; CfCAT was induced in the first 16 hours and and reached the highest point (about 2.9 fold higher than blank group) in 16 hour point after M. luteus challenged; CfCAT was increased gradually, at the 4 hour point, the expression of CfCAT was about 1.2 fold higher than the blank group while had no significance in other time points after challenged by C. lipolytica.The full-length cDNA of CfGPX was of 1290 bp with an ORF of 705bp encoding 235 amino acids with a 24 amino acids signal peptide. BLAST analysis revealed that the predicated amino acid sequence of CfGPX shared high identity with GPX in other organisms. There was one GPX active signature sequence in CfGPX, and the sedieum-cystein was conserved in CfGPX. There was a SElenoCysteine Insertion Sequence in the 3'UTR of CfGPX. Higher-level mRNA expression of CfGPX was detected in the tissues of gonad and muscle. The expression of CfGPX was induced in the first 6 hours and reached the highest point (about 4.0 fold higher than blank group) in 6 hour point then recovered after L. anguillarum challenged; CfGPX was decreased in first 6 hours, and then increased after that in M. luteus challenged group; CfGPX was decreased a little after C. lipolytica challenged. The results indicated that CfSOD, CfCAT and CfGPX were constitutive and inducible acute-phase proteins and could play an important role in the immune responses against microorganisms infection.