Construction Of Bacillus Subtilis Engineering Strain Through Transfering HrpZ_Psgl2

Bacillus subtilis (abbreviated Bs) is one of the most successful species in the application of microbial microbicides. It can inhibit a wide range of pathogens such as fungi, bacteria and other. It has become a research hotspot which genetically modified Bacillus subtilis by molecular biological techniques, and further improve the control effect to the pests.The purpose of this study was to construct Bacillus subtilis expression vector with hrpZPsg12 cloned from Pseudomonas syringae pv.glycinea which causes soybean bacterial blight disease, and transfomed it into the Bs type strain 168, and BcX1 being of biocontrol activity. Physiological and biochemical characteristics as well as biological control activity of both the recombination strains were identified indoor, and it laid the foundation for the further development of new biological pesticides. Specific results were as follows.1. Recombinant strains of BcX1/pHCMC05-hrpZPsg12-gfp and Bs168/pHCMC05-hrpZpsg12-gfp were successfully constructed. hrpZPsg12 and gfp gene fragments were amplified by PCR with the well constructed vector pGEX-4T-hrpZPsg12 and pMD18-gfp, then were connected with vector pMD18-T. The object gene hrpZPsg12 and gfp digested by double enzymies were connected with pHCMC05, respectively to get pHCMC05-hrpZPsg12-gfp plasmid. The recombinant plasmid was transformed into the strain E.coli DH5αfor cloning identification, and the correct vector pHCMC05-hrpZPsg12-gfp were transformed into E.coli BL21 (DE3),Bs168 and BcXl.2. Protein expression conditions for the recombination strain BcX1/pHCMC05-hrpZPsg12-gfp and Bsl68/pHCMC05-hrpZPsg12-gfp were optimized and expressed protein were detected. The optimized expression conditions were:as the concentration of bacteria suspension reached to 0.8 of OD600,IPTG were added to the final concentration of 0.2mM to induce for 8h at 37℃. Fluorescence was detected by fluorescence microscope from expression products and confirmed that gfp-hrpZPsg12 fusion protein had already been expressed. The expression products of recombinated Bs was broken into pieces with ultrasonic method, then precipitated with ammonium sulfate, but no protein were detected by SDS-PAGE.3. The difference between BcXl and BcX1/pWCMC05-hrpZPsg12-gfp in physiological and biochemical characteristics were compared, and changes of inhibitory effect of both of them were detected by plate culture method. The results showed that the physiological and biochemical characteristics in recombination strain BcX1/pHCMC05-hrpZPsg12-gfp and Bsl68/pHCMC05-hrpZPsg12-gfp were not changed and the inhibitory effect was better than the original one.