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

作　　者：卢靖乐[1] 余新燕[1] 侯毅平[1] 王建新[1] 周明国[1] 陈长军[1] 

机构地区：[1]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

出　　处：《中国农业科学》2013年第18期3776-3783,共8页Scientia Agricultura Sinica

基　　金：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)

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3.42×105cfu/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。[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 N J-18 were cured by sodium dodecyl sulfate （SDS） and gyp 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 sucessfuUy transformed into wild strain NJ-18 and C136, a plasmid cured strain from N J-1 8. The transformation efficiency of C 136 could be up to 3.42 ×10^5cfu/μg of plasmid DNA and was about 10-fold of that of N J-18. Compared with N J-18, C 136 had the same antibacterial activity, but its growth was inhibited in a certain extent. N J-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.

关 键 词：NJ-18 质粒消解 化学转化 GFP 定殖 

分 类 号：S512.1[农业科学—作物学]