Carried By Pine Wood Nematode Fluorescence The Pseudomonas Gcm5-1a Peroxidase Study

Pine wilt disease (PWD) is listed as one of the serious diseases to many species of Pinus, which is caused by pine wood nematodes and diffused mostly by Monochamus alternatus. The mechanism of this disease is still unknown since the happening and development of this disease are related to pines, pine wood nematodes, Monochamus alternatus, microorganisms and environmental conditions. In this paper, proteins in culture of Pseudomonas fluorescens GcM5-1A carried by the pinewood nematode, Bursaphelenchus xylophilus were precipitated with ammonium sulfate solutions with different saturation rates and the resulting fractions were used to test lignin peroxidase(LiP) activity and the toxicity to suspension cells of Pinus thunbergii,respectively. We also studied the preliminary purification and characterization of extracellular lignin peroxidase from P. fluorescens GcM5-lA. The results showed that LiP activity mainly existed in 50-70% ammonium sulfate fraction; and this fraction also exhibited relatively strong toxicity besides 0-20% ammonium sulfate fraction. The specific LiP activity was raised from 0.81 U/mg.pr to 21.30 U/mg.pr after two purification steps:ammonium sulfate fractionation and DEAE-Sepharose FF. The optimum pH and temperature of the LiP were about 4.0 and 35℃, respectively. The enzyme was stable at pH 6.0 to 9.0 and at temperatures of 15 to 35℃. The typical peroxidase inhibitors NH2OH·HCl and KCN inhibited 88% and 57% of the enzyme activity, respectively, while the inhibitory effect of NaN3 was as low as 15%. The absorption spectrum of the native enzyme showed a Soret peak at 406 nm. Reduction by 1 mmol/L Na2S2O4 resulted in a marked decrease in absorbance; however, no shift was observed in the Soret peak.A genomic library of the GcM5-1A strain was also constructed and a toxin-producing clone was isolated by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1290 bp encoding a protein of 429 amino acids with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83%,82% and 80% identity with hypothetical protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf 0-1, respectively. The gene encoding full-length protein or without the putative signal peptide was cloned and expressed as a soluble protein in E. coli, and the recombinant proteins were purified to homogeneity by affinity chromatography using a Ni2+ matrix column. Bioassay results showed that the recombinant protein with or without the putative signal peptide was both toxic to suspension cells of P. thunbergii. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease caused by the PWN.