Pseudomonas Fluorescens Gcm5-1a Flagellin To Pine Mechanism Of Protease Inhibitors Against Monochamus Alternatus Application Research

Interaction between flagellin secreted by P. fluorescens GcM5-1A carried by the pine wood nematode (PWN) (Bursaphelenchus xylophilus) and suspension cells of P. thunbergii as well as the lethal effect of flagellin to plant cells were investigated by using immunofluoresces assay, cellular staining, conductivity measurement and electrophoresis analysis. The results showed that there existed a direct interaction between suspension cells of P. thunbergii and flagellin. The cell membrane was shrinkaged, the cytoplasm was concentrated, the nucleus was divided into many micronuclears and the cytoplasmic RNA was degraded in the treated cells. The tinting strength of the treated cell was strengthened accompanied with an increase of culture media conductivity, which implied an increase of the permeability of suspension cell membrane. Electrophoresis analysis of genomic DNA confirmed that DNA breakage happened in the treated cells, but no evident DNA ladder was formed. We supposed that the lethal mode of P. thunbergii cells caused by flagellin is atypical apoptosis.In order to further verify the toxicity of flagellin, a constitutive and secreting expressing plasmid pUC18ompA was constructed. The gene fliC encoding flagellin of Pseudomonas fluorescens Pf-5 was cloned into this plasmid to construct pUC18ompA-fliC. The plasmid was transformed into E.coli BL21 (DE3) to construct engineered bacteria. Bacterium-free seedlings of Pinus thunbergii were inoculated with a mixture of the engineered bacteria and the aseptic PWNs to determine its pathogenicity. The results of inoculation showed that inoculation with a mixture of engineered bacteria and aseptic PWNs also caused wilt of pine seedlings to some extent. The important role of flagellin played in vivo in pathological process was further verified.The management of pine wood disease (PWD) was also investigated preliminary in the research. A cDNA encoding cysteine protease inhibitor (CPI) was isolated from the cDNA library of clamworm Perinereis aibuhitensis Grube. The deduced amino acid sequence analysis showed that the protein had 51%,48%, and 48% identity with Zgc:153129 from Danio rerio, cystatin B from Theromyzon tessulatum and ChainA, stefin B tetramer from Homo sapiens, respectively. The gene was cloned into the intracellular expression vector pET-15b and expressed in Eschechia coli. The recombinant CPI (PA-CPI) was purified by affinity chromatography on Ni-charged resin and ion-exchange chromatography on DEAE-Sepharose FF. The relative molecular mass of PA-CPI was 16 KDa deduced by SDS-PAGE. Activity analysis showed that the recombinant protein could inhibit the proteolytic activity of papain. The cDNA encoding CPI was subcloned into the constitutive and secretive expression vector that was constructed for extracellular expression. Western-blotting analysis results confirmed that the PA-CPI was secreted into the medium. Bioassay demonstrated that E. coli DH5αharboring pUC18ompAcat-CPI showed a significant difference in mortality to the pine sawyer beetle Monochamus alternatus and Asian longhorned beetle Anoplophora glabripennis compared with untransformed E. coli DH5αand control.