枯草芽孢杆菌NJ-18的质粒消解及其在小麦根部的定殖.pdfVIP

中国农业科学 2013,46(18):3776-3783 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2013.18.005 枯草芽孢杆菌NJ-18 的质粒消解及其在小麦根部的定殖 卢靖乐，余新燕，侯毅平，王建新，周明国，陈长军 （南京农业大学植物保护学院/农作物生物灾害综合治理教育部重点实验室，南京 210095 ） 摘要：【目的】研究从土壤中分离出来的具有广谱抗菌活性的枯草芽孢杆菌（Bacillus subtilis）菌株NJ-18 与作物互作关系，并观察外源导入绿色荧光蛋白（green fluorescent protein，GFP）基因的NJ-18 菌株在小麦 根部的定殖能力。【方法】首先使用十二烷基硫酸钠（SDS）消解NJ-18 的质粒，然后采用化学法导入gfp 分子标 记，并采用激光共聚焦显微镜检测该标记菌株在小麦根部的定殖能力。【结果】获得了NJ-18 的质粒消解菌株C136， 并采用化学方法成功将外源基因导入野生型菌株NJ-18 及其质粒消解菌株C136，C136 的转化效率可达到3.42× 5 10 cfu/ μg 质粒DNA，是NJ-18 转化效率的10 倍左右。对比NJ-18 与C136 的生物活性发现，C136 能够保持原有 的抑菌活性；但是C136 的生长曲线第一峰值出现的时间滞后12 h，第一峰值被抑制了29.4%；NJ-18-GFP 能够附 着在病原菌菌体上，导致其菌丝呈现膨大、畸形等；接种7 d 后能够在小麦根部表皮和中柱稳定定殖。【结论】枯 草芽孢杆菌NJ-18 菌株的转化方法宜选用化学法；该菌株能够在小麦根部定殖。 关键词：NJ-18；质粒消解；化学转化；GFP；定殖 Plasmid Elimination of Bacillus subtilis NJ-18 and Its Colonization on Wheat Roots LU Jing-le, YU Xin-yan, HOU Yi-ping, WANG Jian-xin, ZHOU Ming-guo, CHEN Chang-jun (College of Plant Protection, Nanjing Agricultural University/Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing 210095) Abstract: 【Objective 】Bacillus subtilis strain NJ-18, isolated from the soil of oilseed rape field, has a broad spectrum antibacterial activity and a great potential for biocontrol. In order to study the interaction between NJ-18 and plants, NJ-18 was marked with a green fluorescent protein (gfp) gene and its colonization ability on wheat roots was observed with a laser scanning confocal microscope. 【Method 】The plasmids of NJ-18 were cured by sodium dodecyl sulfate (SDS) and gfp was transformed into the strain with a chemi