Purification And Mechanism Study On The Toxins Secreted By Pseudomonas Fluorescens Carried By Pine Wood Nematodes

Pine wilt disease is one kind of lethal disease 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 ,microorganism and environmental conditions. In this disertation, a protein toxin produced by a bacterium strain carried by pine wood nematode was purified and identified, and its toxities to suspension cells and seedlings of Japanese black pine was studied.A protein toxin with a molecular weight of 50 kD was purified from the culture of Luria Broth medium of Pseudomonas fluorescens carried by Bursaphelenchus xylophilus, through ammonium sulfate precipitation , chromatography on DEAE-Sepharose FF column followed by chromatography on Superdex 75 column. N-terminal sequence of the protein was ALSVNTNITS and identified as flagellin. This protein was found to be highly toxic to both suspension cells and seedlings of Pinus thunbergii . Treatment of suspension cells with flagellin could lead to leakages of soluble carbohydrate and free amino acids, as well as the increase of both intra and extracellular peroxidase activities. Peroxidase isoenzymes analysis indicated that a new isoenzyme was produced by the treatment of the toxic flagellin. There exited a direct interaction between suspension cells and flagellin as proved by means of cell immunoflurescense. Therefore, we suspect that flagellins secreted by pine wood nematode-carried Pseudomonas fluorescens might involed in pine wilt disease.Pseudomonas fluorescens carried by pine wood nematodes was cultured in LB,NB and PD mediums, respectively. The cultures free of bacteria were used to analyze their toxic to suspension cells and shoot seedlings of Pinus thunbergii. The results indicated that the cultures of LB and NB were toxic, and LB culture showed the higher toxicity, while almost no toxicity was detected for PD culture. pH values of LB medium affected the toxicity of final culture, the culture with initial pH7.0 was more toxic than that of pH5.0.In order to certify the toxicity of flagellin, the gene encoding flagellin(fliC) of Pseudomonas fluorescens Pf-5 was synthesized in vitro and cloned into pAP3neo . FliC was cutted down by restriction endonucleases NdeI and XhoI, and cloned into pET-15b to construct expression vector pET-15bfliC. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to construct engineering bacterium. Overexpression of flagellin was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on nickel resin column and iron-exchange chromatography on DEAE-Sepharose FF column. Bioassay results showed that the recombinant protein had a similar toxicity to root-cut seedlings and suspension cells of Japanese black pine as wild flagellin.