生防菌枯草芽孢杆菌CQBS03的绿色荧光蛋白基因标记及其在柑橘叶片上的定殖

Construction of Green Fluorescent Protein Gene Tagged Biocontrol Bacteria Bacillus subtilis CQBS03 and Its Colonization on the Citrus Leaves

【目的】利用绿色荧光蛋白(Green fluorescent protein,GFP)基因标记枯草芽孢杆菌生防菌株CQBS03,以研究其在柑橘叶片上的定殖规律。【方法】采用重叠PCR方法将p43启动子和gfp进行融合,并与大肠杆菌-枯草芽孢杆菌穿梭载体pHY300PLK连接构建重组质粒pHY43G,以重组质粒电脉冲转化生防菌株CQBS03,培养后于荧光显微镜下观察并通过SDS-PAGE分析工程菌株GFP的表达情况,然后进行工程菌株对柑橘溃疡病菌的抑菌试验、生长动力学分析、稳定性测试。采用叶片喷雾方法接种,平皿稀释分离培养方法测定CQBS03-pHY43G在柑橘叶片上的定殖情况。【结果】重组菌CQBS03-pHY43G高效表达GFP,电泳后出现约29kDa的特异蛋白条带,外源质粒对宿主菌的生长未带来明显不利影响,质粒稳定性实验表明重组质粒pHY43G经30次传代后的稳定性为55%,室内平板抑菌实验结果显示CQBS03-pHY43G对柑橘溃疡病菌生防效果与出发菌株没有明显差异(P<0.01),CQBS03-pHY43G菌株在叶片喷雾接种后的0—15d,在柑橘叶片上的数量呈现急剧下降趋势,15d后下降速度稍缓,最后保持相对稳定的较低水平(1.73×103cfu·g-1)。【结论】成功地将gfp转入野生型枯草芽孢杆菌生防菌CQBS03中,构建了荧光标记菌株,初步明确了生防菌的定殖规律。

【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（P0.01） 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.