Expression And Function Analysis Of Glutathione S-transferase And Superoxidase Dismutase From Cristaria Plicata

The freshwater mussel Cristaria plicata,which is of great economic importance,is well known as one of “freshwater pearl bivalve” in the aquaculture industry of China.However,the farming of freshwater pearl has been suffering serious problems due to the outbreak of mussel diseases in the cultivation process.Thus,it is crucial for diseases management and development of sustainable mussel culture and pearl production to research the immunity of freshwater mussel.Glutathione-S-transferases(GST5s)are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione and play an important role in protecting organisms from the toxicity of reactive oxygen species.In this study,an alpha-level GST5(CpGST5)cDNA sequence was cloned from the freshwater bivalve Cristaria plicata.The full-length cDNA of CpGST5 is 1185 bp,and the open reading frame is 669 bp,encoding 222 amino acid residues.Protein structure analysis indicated that the mature peptide of CpGST5 includes two typical domains,the conserved domain GST_N and the conserved domain GST_C.N_terminal contains a GSH binding site(G site).The C_terminal contains a site for the substrate binding pocket(H site).Real-time quantitative PCR results showed that CpMnSOD mRNA was constitutively expressed in each tissue.The CpGST5 gene has the highest expression in hepatopancreas and the lowest expression in the mantle.After stimulation with microcystins,the expression level of CpGST5 in hepatopancreas and hemocytes showed a markedly increasing trend.Recombinant CpGST5 was expressed in the form of inclusion bodies in E.coli DE3.Manganese superoxide dismutase(MnSOD)is a sort of important metalloenzyme that catalyze ROS to form harmless molecular oxygen and hydrogen peroxide in the organisms.In this study,MnSOD cDNA of C.plicata,designated as CpMnSOD(accession no.MK465057),was cloned from hemocytes using degenerate primers by the rapid amplification of cDNA ends PCR.The full-length cDNA of MnSOD was of 1096 bp with a 672 bp open reading frame encoding 223 amino acids.The deduced amino acid sequence contained a mitochondrial-targeting sequence(MTS)of 18 amino acids in the N-terminus,and four conserved amino acids for manganese binding(H49,H97,D182,H186).Screening of CpMnSOD 5'-flanking region revealed the presence of several important transcription factor binding sites that potentially govern the MnSOD expression.These findings conjointly contribute to expanding our understanding regarding the mussel MnSODs.CpMnSOD showed a high level(65-73%)of sequence similarity to MnSODs from other species.The results of Real-time quantitative PCR revealed that CpMnSOD mRNA was constitutively expressed in tissues.The highest expression level was in hepatopancreas,followed by muscle,mantle and gill,and the lowest expression level was in hemocytes.After microcystin challenge,the expression levels of CpMnSOD mRNA were up-regulated in hemocytes and hepatopancreas.The cDNA of CpMnSOD was cloned into the plasmid pColdI-ZZ,and the recombinant protein was expressed in Escherichia coli BL21(DE3).The enzyme stability assay showed that the purified CpMnSOD protein maintained more than 80% enzyme activity at temperature up to 70°C,at pH 2.0-10.0,and was resistant to 8 mol/L urea or 8% SDS.