Function Identification Of The Insecticidal Gene Hs487 From Serratia Nematodiphila R187-2

Symbiotic bacterial strains of the entomopathogenic nematode-bacterium complex play an important role in killing insect.These symbiotic bacterial strains,usually harbored in the gut of their nematode symbionts,belong to three genera of the family Enterobacteriaceae,Xenorhabdus,Photorhabdus and Serratia,which have symbiotic relationship with nematodes of families Steinernematidae,Heterorhabditidae and the genus Heterorhabditidoides,respectively.Our previous studies isolated a highly pathogenic bacterial strain,Serratia nematodiphila R187-2,from the gut of Heterorhabditidoides rugaoensis and found that the strain R187-2 had significantly insecticidal activity against the fifth-instar larvae of Galleria mellonell.The major insecticidal active ingredient of this strain was its extracellular toxin protein HS487.To explore further the characteristics of the insecticidal toxin HS487,we established successfully a prokaryotic expression system of the toxin and its partial-deficiency mutant strain.Main methods and results in this study are as follows.The total gene fragment of HS487 was obtained by PCR method,and used for constructing its prokaryotic expression system with the pET prokaryotic expression system in Escherichia coli BL21(DE3).The recombinant protein of hs487 was mainly expressed in the form of inclusion body.The optimal induction conditions.for the protein were treated with 0.2 mM IPTG for 3 h at 30℃.The recombinant toxin,existed in the form of inclusion body,was purified and renatured by subsequent steps of washing,urea solution,Ni-chelating affinity chromatography,gradient dialysis,tracked by SDS-PAGE.Value of LD50 of the recombined HS487 against the fifth-instar larvae of Galleria mellonella was 2.71(2.29-3.16)ng-mg-1" by injected method,which indicated that inclusion body protein renaturation was successful and had a strong insecticidal activity.In order to constructed the partial-deficiency mutant strain of the insecticidal hs487,hs487s,a 460bp fragment of hs487,was cloned and inserted into the plasmid pUTKml to establish the recombinant plasmid pUTKm-hs487s,which was then transformed into E.coli S17-1(λpir).The mutant strain HS487S was constructed successfully by the parent hybridization method.Bacterial strains of mutant and wild type of R187-2 were used to infected two immune-deficiency mutants and the wild stain of Drosophila mellonagaster.Expression level of the two antibacterial peptide,Diptericin arid Drosomycin,in these Drosophila strains were detected by fluorescence·quantitative PCR method.Results showed that the expression level of the two peptides in Drosophila infected by the mutant HS487S were relatively lower than R187-2 infection,which confirmed that the immune response of Drosophila infected by the mutant HS487S was relatively slower than R187-2 infection.Correspondingly,the ability of the mutant strain HS487S to kill insects was significantly reduced,so that individuals of Drosophila can survive longer than R187-2 infection.Insecticidal activity of R187-2 was significantly decreased after the insecticidal genes hs487 were broken.These results proved that the insecticidal genes hs487 is the key virulence factors in Serratia nematodiphila R187-2.