Expression Of The FtsZ From Xanthomonas Oryzae Pv. Oryzaein Vitro And Effect Of The Antiobiotics From Micromonospora Carbonacea JXNU-1 On Its Gtpase Activity

Micromonospora carbonacea JXNU-1 is an Actinomyces with broad-spectrum antibacterial activity, isolated from soil. The sole component of antibiotics from Micromonospora carbonacea JXNU-1 exhibits antibacterial activity against to common G+ and G-, however its antibacterial mechanism is not clear. Therefore, the effect of antibiotics from M. carbonacea JXNU-1 on the FtsZ protein from Xanthomonas oryzae pv.oryzae, a kind of bacteria being the cause of bacterial blight of rice which is great harmful to rice, was studied in this study, the specific contents and results are as follows:(1) The ftsZ gene was amplified from X. oryzae pv.oryzae by nested PCR, and recombinant plasmid pET-22b-ftsZ was constructed and transformed to E.coli BL21. The clony fragment was identificatified by PCR screening, Nde I/Xho I digestion and DNA sequencing, the positive clones were induced by IPTG for expression. the fusion protein was purified through Ni-NTA Resin, and identified by SDS-PAGE and Western blotting. Results showed that the recombinant plasmid pET-22b-ftsZ was constructed successfully, the FtsZ protein was expressed in recombined E. coli BL21 induced by IPTG, and purified through Ni-NTA Resin by electrophoretic purity.(2) The expression conditions for recombinant E. coli BL21 (pET-22b-ftsZ) were optimized by single factor method. Results showed that the optimized conditions was temperature being 37℃, bacteria concentration being 0.8 (OD600), IPTG concentration being 1.0 mmol/L and induction time being 3h.(3) The structure and physicochemical properties of FtsZ protein was analyzed in ExPASy. The results demonstrated that protein FtsZ is stable and hydrophilic, containing 414 amino-acid residues. Among them, the fragment from 15th to 187th amino-acid residue determines the activity of GTPase and the fragment of GGGTGTG represent the common combining site of GTP, As for the secondary structure, the percentages of spiral structure and folding structure are 30.4% and 11.8% respectively.(4) The characteristics of GTPase activity of FtsZ protein was analyzed by the method of enzymology. The optimal conditions for the GTPase activity of FtsZ protein are the temperature of 50℃, the pH value of 7.0 and with the presence of Mg2+, the value of Km is 5.78 mmol/L.(5) The growth of X. oryzae pv.oryzae can be inhibited by antibiotics from M. carbonacea JXNU-1. The filamentous cells of X. oryzae pv.oryzae were seen when inhibited by antibiotics at the concentration of MIC, and the GTPase activity of FtsZ protein was completely inhibited by antibiotics.All results above expounded the antimicrobial mechanism of antibiotics from M. carbonacea JXNU-1, that is the antibiotics inhibit the GTPase activity of FtsZ protein, and then inhibit the cell division of X. oryzae pv. Oryzae.