Study On Antagonistic Protein’s Mechanism Of Action And Active Domains Of ChiA Gene From Brevisbacillus Laterosporus Strain B8

Brevibacillus lateroporus strain B8 which isolated from the rhizosphere soil around tobacco plants with great biological activity. The fermentation solution of strain B8 showed inhibiting effects on Phytophthora capsici, Rhizoclonia solani and other pathogens. Chitinase and antagonistic protein could be produced by this strain which molecular weights were 77.6 kDa and 11.2kDa, respectively. Strain B8 has strong bio-control potential and developmental prospects considering its strong inhibition, stable inhibiting activity and wide antimicrobial spectrum. Action mechanism of antagonistic protein from B.laterosporus strain B8 and the active domain of its chiA gene were studied in this article. The results were as follows:1. Inhibiting effects and inhibiting mechanism of antagonistic protein from B.laterosporus strain B8 on R.solani and P.capsici were studied. The antagonistic protein was separated and purified by ammonium sulfate precipitation method, dialysis desalting, TLC, Pharmadex LH-20 column chromatography and HPLC. The results indicated that different concentrations of inhibiting effects of antagonistic protein were different, the inhibition rate enhanced with the increase of the antagonistic protein concentration. R.solani and P.capsici mycelia were inhibited effectively treated with 5.63mg·mL-1antagonistic protein from B.laterosporus strain B8 which was the best concentration. Mycelias were observed microscopically, swellings and broken of mycelia, cytoplasm exosmosis, cell wall degradation, at last the hyphae disintegrated after processed by antagonistic protein; was observed excessive branching of mycelia, malformation of mycelia tips and protoplasm cavitations, at last broke into small peaces. Protoplast vesicles and hyphae fracture were found in P.capsici, while swelled hypha, fragmented mycelium and cell walls digestion were observed in R.solani.2. Resistance mechanism to R.solani and P.capsici, the growth promoting effect to pepper seed were studied. Sweet peppers were treated by antagonistic protein and inoculated with R.solani and P.capsici 3 days later. The results indicated that disease resistance of R.solani and P.capsici were induced by the antagonistic protein, with the resistance efficiency over 80%, and control effect could remain more than 12 days after the treatment. Pathogenesis-related proteins (PRs) were induced in pepper plants from the 4th day after treatment with the analysis of molecular weight of protein by SDS-PAGE electrophoresis, which could induce plant resistance against R.solani and P.capsici. The results of 6 days’ protein maps was the same as 4 days’. Therefore, PR proteins induced in peppers after 4 days and 6 days treated with the antagonistic protein, pepper plants showed resistance against the infection of R.solani and P.capsici as a result. Furthermore, the antagonistic protein of B8 could prompte sweet pepper seeds germination and growth.3. Based on the analysis results of B.lateroporus strain B8 chiA protein structure domains, strain B8 chiA gene and conservative domains were induced to structure, the the activity structure domain of B8 chiA gene was confirmed combining the activity analysis of protein structure domain. A 38aa signal peptide of N-terminal, a 410aa O-glycosyl hydrolase structural domain and a 138aa chitin-binding domain were found in the coding region by strainB8 chiA protein structure domain analysis results. chiA gene and coding region vectors were structured respectively and were identified after screening by PCR, double enzyme digestion with EcoR I and Xho I, sequence analysis. The recombinant expression vectors pET-28a-chiA-f, pET-28a-chiA-d and pET-28a-chiA-c of strain B8 chiA gene were constructed. Recombinant expression vectors were successfully transformed into E. coli BL21 and protein was induced and expressed by IPTG. Recombinant expressed protein was separated and purified by Ni-NTA affinity purification. Inhibiting activities and chitinase activities of recombinant expressed protein were measured by oxford cup method. The results indicated that chiA gene and O-glycosyl hydrolase structural domain recombinant proteins inhibited R.solani obviously, which formed transparent zones on the chitin tablets; chitin-binding domain recombinant proteins inhibited R.solani ineffectively, which didn’t form transparent zones on the chitin tablets, thus verifying O-glycosyl hydrolase structural domain was the active domain of B.lateroporus strainB8 chiA gene.