枯草芽孢杆菌NJ-18的质粒消解及其在小麦根部的定殖

doi:10.3864/j.issn.0578-1752.2013.18.005

中国农业科学 ›› 2013, Vol. 46 ›› Issue (18): 3776-3783.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

收稿日期:2013-03-01 出版日期:2013-09-15 发布日期:2013-05-22
通讯作者: 通信作者陈长军，Tel：025-84395641；E-mail：Changjun-chen@njau.edu.cn
作者简介:卢靖乐，E-mail：lusanle@163.com
基金资助:
国家“973”（2009CB118906，2012CB114000）、国家“863”（2012AA101502）、国家自然科学基金项目（30971891，31171880）、国家科技支撑计划项目（2012BAD19B01）、农业部公益性（农业）科研专项（200903052，201103016）

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

Received:2013-03-01 Online:2013-09-15 Published:2013-05-22

摘要/Abstract

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3.42×105 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

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 chemical method. Colonization ability of NJ-18 on wheat roots was observed with a laser scanning confocal microscope. 【Result】 By a chemical method, gfp was sucessfully transformed into wild strain NJ-18 and C136, a plasmid cured strain from NJ-18. The transformation efficiency of C136 could be up to 3.42×105 cfu/μg of plasmid DNA and was about 10-fold of that of NJ-18. Compared with NJ-18, C136 had the same antibacterial activity, but its growth was inhibited in a certain extent. NJ-18 could attach on the pathogen microbial cells, resulting in mycelium enlargement and malformations. NJ-18 was able to colonize in the phloem and xylem of wheat roots on 7 days after inoculation. 【Conclusion】 The chemical transformation method is suitalbe for strain NJ-18. NJ-18 can colonize on wheat roots.

Key words: NJ-18 , plasmid-cured , chemical transformation , GFP , colonization

卢靖乐, 余新燕, 侯毅平, 王建新, 周明国, 陈长军. 枯草芽孢杆菌NJ-18的质粒消解及其在小麦根部的定殖[J]. 中国农业科学, 2013, 46(18): 3776-3783.

LU Jing-Le, YU Xin-Yan, HOU Yi-Ping, WANG Jian-Xin, ZHOU Ming-Guo, CHEN Chang-Jun. Plasmid Elimination of Bacillus subtilis NJ-18 and Its Colonization on Wheat Roots[J]. Scientia Agricultura Sinica, 2013, 46(18): 3776-3783.

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枯草芽孢杆菌NJ-18的质粒消解及其在小麦根部的定殖

Plasmid Elimination of Bacillus subtilis NJ-18 and Its Colonization on Wheat Roots

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