Cloning And Activity Of Peroxidase Gene Of Pseudomonas Fluorescens GcM5-1A Peroxide Reductase And

Peroxiredoxins (Prxs) are enzymatic antioxidants widely distributed in biological kingdoms, which constitute a family of heme-free peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. In this paper, an open reading frame of639bp, which encoded a protein of213amino acid residues, was cloned from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode. Amino acid sequence alignment showed that the encoded protein shared a similarity of99%,97%and97%identity with the thiol-specific antioxidant protein LsfA of P. fluorescens Q2-87, the peroxiredoxin of Pseudomonas sp. GM17and1-Cys peroxiredoxin of P. fluorescens PfO-1, respectively. The ORF was cloned into expressing vector pET-15b to construct recombinant plasmid pET-15b-1-Cys-Prx which was introduced into in Escherichia coli BL21(DE3). Overexpression of a27kDa protein was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni2+matrix column. Non-reducing SDS-PAGE analysis indicated that part of the recombinant appeared in dimer form. Bioassay results showed that purified recombinant protein had both peroxidase and thioredoxin activity. E. coli harboring the ORF showed tolerance to hydrogen peroxide stress.In addition, a peroxidase cDNA containing opening reading frame of981bp and encoding a peptide of326amino acid residues was obtained from callus cells of Pinus thunbergii using RT-PCR. The open reading frame was cloned into pET-15b to construct recombinant plasmid pET-15b-POD. This recombinant plasmid was introduced into Escherichia coli BL21(DE3) to construct engineer bacteria and expression of a38kDa recombinant peroxidase was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni2+matrix column. Bioinformation analysis showed that the protein had typical structures of plant peroxidase and possessed all the conserved active sites. Bioassay results showed that purified recombinant protein had peroxidase activity. This study laid a good foundation for further research on the resistant mechanism of pine wilt disease.