CLONING AND PROKARYOTIC EXPRESSION PEROXIREDOXIN 6 GENE IN CRISTARIA PLICATA

Peroxiredoxin 6 has glutathione peroxidase and phospholipid enzyme A2 double activity and plays an important role in antioxidant protection and metabolism of lung surface-active material. Cristaria plicata is one of the most important freshwater mussels for pearl production in China. In this study, a Prx6 homologue-CpPrx6 in Cristaria plicata, was cloned by utilizing reverse transcriptase polymerase chain reaction and rapid amplification of the cDNA ends. A phylogenetic tree was constructed based on the amino acid sequence of CpPrx6 using neighbor-joining method and MEGA 4.1 software. The three-dimensional structure of CpPrx6 was constructed using the Swiss-model. The expression patterns, both in tissues and towards microorganism A. hydrophila stimulation, were then characterized by real-time fluorescence quantitative PCR. The results showed that the full length cDNA of CpPrx6 contained a 5'- untranslated region (UTR) of 71 bp, an open reading frame of 657 bp that encoded 218 amino acids, and a 3'-UTR of 889 bp with a 29 bp polyadenylic acid tail. The predicted molecular mass and estimated isoelectric point (pI) of CpPrx6 were 24.24 kD and 6.33, respectively. No signal peptide, the transmembrane region, mitochondria, lysosomes, peroxisomes and nuclei localization sequence were found and contained 1 potential N-linked glycosylation site (N9FTA) in CpPrx6, which suggested that it is a cytosolic protein. The deduced amino acid sequence of CpPrx6 shared 70%-72% overall similarity with known Molluscan Prx6. The conserved peroxidase catalytic center PVCTTE and a lipase motif GKSWA were observed in the sequence, the tertiary structures of CpPrx6 contained 6 -helixes and 12 -sheets, the catalytic center PVCTTE located in fifth -helix, three conserved residues His22, Asp134 and Ser28 together formed the catalytic center of the phospholipase A2, indicating that it was a member of 1-Cys Prx. Phylogenetic analyses showed that CpPrx6 sequence clustered together with the other Molluscan Prx6 sequences. The mRNA transcript of CpPrx6 was detected in most C. plicata organs, such as haemocyte, mantle, adductor muscle, hepatopancreas and gill, with the highest expression level found in gill. After injection of Aeromonas hydrophilai, the expression levels of CpPrx6 significantly increased in hepatopancreas at 6h and 12h, in haemocytes and gills at 12h (6h, P0.05; 12h, P0.01), respectively. The expression levels then returned to normal at 24h. This suggested that CpPrx6 perhaps played an important role in resistance to oxidative stress and basic immunization of C. plicata. According to the ORF, designed site (KpnI and EcoRI) primers, the CpPrx6 gene was subcloned into a vector pET-32a (+) and transformed into Escherichia coli BL21 (DE3), the constructed recombinant expression plasmids were induced by the chemical inducer IPTG. The expression products were analyzed by SDS-PAGE. The results indicated that recombination CpPrx6 was successfully expressed in E. coli. The expressed protein had a molecular weight of 40 kD.