Gene Cloning And Activity Study Of Pseudomonas Fluorescens GcM5-1A Hemolysin

A pair of special primers were designed according to the gene encoding hypothetical hemolysin of Pseudomonas fluorescens GcM5-1A carried by Busaphelenchus xylophilus.This gene was amplified by PCR using the designed primers and the genomic DNA of P.fluorescens GcM5-1A as the template.The PCR product was cloned into expression vector pET-15 b to construct pET-15b-Hly,followed by transforming this recombinant plasmid to E.coli BL21(DE3)to construct engineering bacteria.Engineered bacteria produced one more protein than E.coli BL21(DE3)without expression plasmid after IPTG induction was applied.This protein is about 26 kDa.The result was consistent with the predictions.Hypothetical hemolysin in engineering bacteria was expressed.The recombinant protein was purified by Ni-Sepharose 6(FF)affinity chromatography.Red cell lysis test indicated that the recombinant protein could lyse chicken erythrocytes,which illustrated that the gene cloned in this paper indeed coded hemolysin.Hemolysin can be divided into three categories: enzymes,pore formation moleculars and surfactants.In order to investigate the hemolytic mechanism of recombinant hemolysin of P.fluorescens GcM5-1A,we conducted the experiments of osmotic protection,the effect of metal ions and enzyme inhibitors on hemolytic activity of hemolysin and heat treatment at 100?.The results showed that metal ions and enzyme inhibitors did not promote or inhibit the recombinant hemolytic activity of P.fluorescens GcM5-1A,hemolysin lost its hemolytic activity after treatment at 100?,and PEGs could inhibit hemolysin activity.All these showed that recombinant hemolysin of P.fluorescens GcM5-1A was one of the pore forming toxins.The toxicity of the recombinant hemolysin to Pinus thunbergii was studied.The results showed that the the recombinant hemolysin could the death of pine seedlings,while sterile water treatment caused no wilting phenomenon.The mortality of black pine seedlings treated with hemolysin increased gradually with the increase of hemolysin concentration.Seedlings of P.thunbergii began to wilt when they were treated with 20 ?g/ mL hemolysin for 4 d,and all of the pine seedlings died when they were treated with100 ?g / mL hemolysin.when callus cells of P.thunbergii were treated with 60 ?g / mL hemolysin at 4? for 4 d,the survival rate of callus cells was 21.41% lower than that of the control callus cells.Hemolysin could cause callus cells of P.thunbergii to die whencallus cells were treated with 60 ?g / mL hemolysin for 24 h at 27?.All the results showed that P.fluorescens GcM5-1A could synthesize hemolysin,and the recombinant hemolysin had a lethal activity against the black pine seedlings and callus cells.The study laid the foundation for the further study of the roles of bacteria associated with B.xylophilus in pine wilt disease.