枯草芽孢杆菌BSD-2的GFP标记及其在黄瓜上的定殖研究

doi:10.7668/hbnxb.2016.04.018

摘要/Abstract

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pGFP4412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。

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

Abstract: 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 pGFP4412 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.

Key words: Bacillus subtilis, GFP, Fusarium oxysporum, Botrytis cinerea, Inhibitory activity

中图分类号:

Q78
S436.421

郝慧娟, 刘洪伟, 尹淑丽, 刘倩倩, 张丽萍, 宋水山. 枯草芽孢杆菌BSD-2的GFP标记及其在黄瓜上的定殖研究[J]. 华北农学报, 2016, 31(4): 106-111. doi: 10.7668/hbnxb.2016.04.018.

HAO Huijuan, LIU Hongwei, YIN Shuli, LIU Qianqian, ZHANG Liping, SONG Shuishan. GFP-Marking and Colonization on Cucumber of Bacillus subtilis BSD-2[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(4): 106-111. doi: 10.7668/hbnxb.2016.04.018.

http://www.hbnxb.net/CN/Y2016/V31/I4/106

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