Molecular Cloning,Expression And Activity Analysis Of 2-Cys Peroxiredoxin From The Yellow River Cyprinus Carpio

The common carp is an important aquatic economic animal in our country. Yellow river common carp is one of the important species in common carp. In the recent years, the influence of human activities, such as overfishing, indiscriminate fishing, stream pollution,natural drying up of Yellow River and so on, destroyed the ecological balance of Yellow river basin. Total resource amounts of the Yellow river carp have declined sharply. With the enlargement of carp farming, the disease occurred frequently, which seriously restricted the healthy development of the Yellow River carp farming industry. More and more attention has been paid for the research of the disease resistance of common carp. Peroxiredoxin exists in a variety of organisms, which is a member of the antioxidase family. They could maintain the intracellular redox balance by eliminating hydrogen peroxide and alkyl hydrogen peroxide. In this study, Prx3 and Prx4 of typical 2-Cys Prx genes was cloned and characterized. Better understanding of the two antioxidant proteins will be beneficial to provide an effective basis for the study of the mechanism of disease resistance in fish.According to the whole genome sequence information of the Common carp provided in NCBI, the Prx3 and Prx4 were cloned by RT-PCR method. The full-length genome sequence of Prx3 is 2816 bp, containing a 5,UTR(Untranslated Regions, UTR) of 196 bp, an open reading frame(Open Reading Frame, ORF) of 753 bp, encoding 250 amino acids. The protein deduced had a molecular mass of 27.01 kDa and a theoretical isoelectric point is 8.25. Prx3 secondary structure contains 29.60% α-helix and 7.20% β-turn. The full-length genome sequence of Prx4 is 3475 bp, an ORF of 783 bp, encoding 260 amino acids. The protein deduced had a molecular mass of 29.11 kDa and a theoretical isoelectric point is 5.98. Prx4 secondary structure contains 31.15% α-helix and 10.77% β-turn. The homology analysis of Prx3 and Prx4 showed that common carp and the other fish have contained two common conserved catalytic center "FTFVCPTEI" and "GEVCPA". Blast and phylogenetic tree showed that the amino acids sequence of common carp Prx3 and Prx4 had the higestsimilarity with zebrafish, platyfish and stickleback. Expression of Prx3 and Prx4 in 12 tissues of Yellow river common carp was determined by the RT-PCR, Prx3 expressed highest in kidney, followed by muscle and lowest in fin, Prx4 expressed highest in kidney, followed by gonad and lowest in airbladder. After challenged with Aeromonas hydrophil, the change of expression was analyzed by extracting the liver, spleen and head kidney, which indicated Prx3 and Prx4 of the three tissues had significant changes in different time periods. In spleen, the expression levels of Prx3 and Prx4 increased obviously at 6h after infection, in kidney, the expression levels of Prx3 and Prx4 declined obviously at 6h after infection. In liver, the expression levels of Prx3 and Prx4 increased obviously at 6h after infection. All the results showed Prx3 and Prx4 had a strong response when Yellow river common carp was infected with the pathogen.After mature peptide of Prx4 gene was amplified, the confirmed recombinant plasmid was digested with Sal I and EcoR I. Verified by enzyme digestion and DNA sequencing, the target gene was correctly inserted into the pMAL-c5 x. The recombinant expression plasmid pMAL-c5x-Prx4 was transformed in E.coli BL21. SDS-PAGE test showed that Prx4 was expressed in E.coli BL21 in the form of soluble recombinant protein and molecular mass of the recombinant Prx4 was about 72 kDa. Western blotting showed that Prx4 protein was expressed in the liver, gill, eye and other tissues. The recombinant Prx4 purified from Dextrin Beads 6FF was found to restore H2O2 in the presence of dithiothreitol. In addition, the disc diffusion assay was further performed to evaluate the recombinant protein Prx4 was able to confer tolerance to oxidative stress in vivo. These results provided the expression characteristic and antioxidant activity of Prx3 and Prx4 in the Yellow river common carp.