枯草芽孢杆菌BSD-2的GFP标记及其在黄瓜上的定殖研究  

GFP-Marking and Colonization on Cucumber of Bacillus subtilis BSD-2

作　　者：郝慧娟[1,2,3] 刘洪伟[2,3] 尹淑丽[2,3] 刘倩倩[2,3] 张丽萍[2,3] 宋水山[2,3] HAO Huijuan LIU Hongwei YIN Shuli LIU Qianqian ZHANG Liping SONG Shuishan(College of Chemical Industry,Hebei University of Technology,Tianjin 300130,China Biology Institute, Hebei Academy of Sciences, Shijiazhuang 050081, China Hebei Engineering and Technology Center of Microbiological Control on Main Crop Disease,Shijiazhuang 050081 ,China)

机构地区：[1]河北工业大学化工学院,天津300130 [2]河北省科学院生物研究所,河北石家庄050081 [3]河北省主要农作物病害微生物控制工程技术研究中心,河北石家庄050081

出　　处：《华北农学报》2016年第4期106-111,共6页Acta Agriculturae Boreali-Sinica

基　　金：河北省重点基础研究项目(13966503D);河北省省级省校科技合作开发资金支持项目(Y-15);河北省科学院重点项目(2015302)

摘　　要：为研究枯草芽孢杆菌BSD-2在黄瓜植株上的定殖情况,采用稍加改进的电击转化方法将含有GFP基因的p GFP4412质粒导入枯草芽孢杆菌BSD-2菌株中,并测定了其生长曲线、质粒遗传稳定性及其对枯萎病菌和灰霉病菌的抑制作用。结果表明,成功获得具有绿色荧光的菌株BSD-2-GFP;标记菌株的生长趋势与野生型菌株基本一致;在无选择压力条件下连续培养56 h,菌株BSD-2-GFP的遗传稳定性为86%;其对植物枯萎病菌和灰霉病菌具有很强的抑制作用,与野生型菌株相当。通过荧光显微镜观察发现,标记菌株处理24 h后即可在黄瓜根内部发现,5 d后可在叶脉发现,且50 d后仍可在叶脉观测到。结果表明,菌株BSD-2-GFP可以很好地在黄瓜体内定殖,从而阻止病原菌的侵入。For the purpose of exploring the colonization on the cucumber,the Bacillus subtilis BSD-2 had remarkable control effect on the Botrytis cinerea. In this study,the plasmid of p GFP4412 that contains green fluorescent protein gene（ GFP） was transformed into the Bacillus subtilis BSD-2 by modified electroporation. The plasmid stability,growth curve and the inhibition activity of GFP-labeled strains were measured. The results showed that the GFP-tagged strain could emit green fluorescence successfully. The tagged strain nearly had the same trend with the wild type strain in growth. The stability of GFP-marked in engineering B. subtilis BSD-2 strain was 86% after transference of culture 56 hours continuously without selective pressure. Inhibition activity showed that the GFP-marked strain exhibited the comparable ability as the wild type strain to inhibit Fusarium oxysporum and Botrytis cinerea.Observation by fluorescence microscope indicated that the GFP-tagged BSD-2 could colonize on the root tissue after inoculated 24 hours. It could be seen on the leaf veins after five days. It still could be observed on the leaf veins after 50 days. All of these data indicated that the GFP-marked strain could colonize on the cucumber so properly that stopped pathogens to invade the plant.

关 键 词：枯草芽孢杆菌 绿色荧光蛋白 枯萎病 灰霉病 抑菌作用 

分 类 号：Q78[生物学—分子生物学] S436.421[农业科学—农业昆虫与害虫防治]