Cloning, Expression And Characterization Of Superoxide Dismutase Gene Of Pseudomonas Fluorescens

Superoxide dismutase containing?SOD?is a large number of antioxidant enzymes found widely in the organisms.Its biological function is mainly related to scavenging of superoxide free radicals produced by the organisms because of aerobic metabolism and protection of cells from injuring of reactive oxygen species.SOD catalyzes the dismutation reaction of superoxide anion,which disproporte O₂^-.to H₂O₂ and O_2.Pseudomonas fluorescens GcM5-1A is a strain of bacterium carried by pine wood nematode,and previous studies indicated this strain has played some roles in pathogenesis process of pine wilt disease coordinated with pine wood nematode.In this study,an open reading frame?ORF?encoding superoxide dismutase?SOD?was cloned from genomic DNA of P.fluorescens GcM5-1A by PCR technology.The sequence analysis showed that the ORF was 654 bp in length,encoding 217 amino acid residue.The recombinant expression plasmid pET-15b-SOD was constructed by cloning the SOD gene of P.fluorescens GcM5-1A into the expression vector pET-15b.The recombinant plasmid was transformed into Escherichia coli BL21?DE3?to construct engineering bactium.SDS-PAGE analysis showed that the recombinant SOD with a relative molecular weight of 28kDa was highly expressed in engineering bacterium induced by isopropyl-beta-D glucosinolates?IPTG?after expanded culture.The recombinant SOD appeared mainly in inclusion bodies.After the recombinant SOD in inclusion bodies was dissolved in urea solution and refolded by dilution,it was purified by Ni²⁺resin affinity chromatography.The purified recombinant SOD was used to assess its enzyme activity.The activity of superoxide dismutase SOD under different temperatures and pH values was also studied.The results showed that the optimal temperature of the SOD was 50?and the optimal pH was pH8.0.The SOD showed a good thermal stability and relative was relatively stable in neutral and weak basic conditions,and E.coli expressing recombinant SOD showed an obvious tolerance to paraquat.In a summary,the gene of superoxide dismutase SOD was cloned from P.fluorescens GcM5-1A,a method for preparing the recombinant SOD was established,and characters of the recombinant SOD were invesitigated.This study lays a foundation for elucidating the roles of bacteria associated with pine wood nematode,and offers some references for application of SOD.