Abstract:

【Objective】 The citrus canker biocontrol bacterium Bacillus subtilis CQBS03 was labeled with green fluorescent protein gene to investigate the colonization of the bacterium on the citrus leaves. 【Method】 The gene promoter p43 and green fluorescent protein (gfp) were connected by overlapping PCR, then the fusion gene p43-gfp was inserted into shuttle vector pHY300PLK and transferred into original strain CQBS03 by electroporation. The expression of GFP was visualized under the fluorescent microscope, and analized by SDS-PAGE. Antibacterial (Xcc01) activities of the engineering strain, dynamic analysis and stability were tested. And the re-colonization on the citrus was examined by the bacteria isolation and culture after leaf acupuncturing inoculation sprayed on leaves of citrus. 【Result】 GFP was expressed efficiently and the protein about 29 kDa was detected by SDS-PAGE in engineering strain CQBS03-pHY43G. Heterogenous plasmid did not lead to significant adverse effects on the engineering strain. The stability of engineering strain was 55% after 30 generations. Antibacterial activities testing proved that there was no obvious difference (P<0.01) between original strain CQBS03 and engineered strain. The population of strain CQBS03-pHY43G on leaves of citrus decreased sharply in the first 15 days after sprayed, then declined slowly after 15th day and remained constant at a lower level (1.73×103 cfu?g-1). 【Conclusion】 The gene of gfp was inserted into Bacillus subtilis CQBS03 successfully and GFP-tagged strain was obtained, the colonizing patterns of bio-control bacterium were preliminarily elucidated.

Key words: promoter of p43, gfp labeling, colonization, Bacillus subtilis, Xanthomonas citri ssp. citri