枯草芽孢杆菌NCD-2菌株的高效电击转化  

High-efficiency Electro-transformation of Bacillus subtilis Strain NCD-2

作　　者：王培培[1,2] 郭庆港[2] 李社增[2] 鹿秀云[2] 李宝庆[2] 张莉萍 马平[2] 董金皋[1] 

机构地区：[1]河北农业大学植物保护学院,保定071000 [2]河北省农林科学院植物保护研究所/河北省农业有害生物综合防治工程技术研究中心,保定071000 [3]河北省张家口市植物保护植物检疫站,张家口075000

出　　处：《中国生物防治学报》2011年第3期368-372,共5页Chinese Journal of Biological Control

基　　金：国家自然科学基金(30900962);现代农业产业技术体系建设项目(nycytx-06)

摘　　要：本研究对影响枯草芽孢杆菌Bacillus subtilisNCD-2菌株遗传转化的因素进行摸索并对其转化条件进行优化,建立了该菌株的高效电击转化体系。通过比较5个不同大小和携带不同来源复制子的质粒对其转化效率的影响,发现质粒大小与NCD-2菌株的电击转化效率之间没有相关性;而携带来源于Bacillus coagu-lans复制子的质粒pNW33N时,NCD-2菌株的电击转化效率最高,达到3.53×104cfu.μg-1DNA,说明复制子来源是影响转化效率的一个重要因素。采用pNW33N质粒在不同电阻、电场强度及培养时间下的电击转化效率的研究结果表明,该菌株转接到生长培养基后,37℃、150r.min-1培养3.5h,在电阻值200Ω、电场强度14.0kV.cm-1的条件下转化效率最高,达到6.07×104cfu.μg-1DNA。优化后的转化体系为顺利开展枯草芽孢杆菌NCD-2菌株的分子操作奠定了基础。Transformation efficiency of Bacillus subtilis stain NCD-2 was compared when the transformation was performed by using 5 different plasmids with various sizes and replication origins.Results showed that the replication origin of plasmid had a significant effect on the transformation efficiency,but not the plasmid size.For example,plasmid pNW33N carried the replication from B.coagulans showed the highest transformation efficiency by 3.53×104 cfu·μg-1 DNA.Other parameters affecting the transformation efficiency,including electric resistances,electric field strengths and cell growth phases,were also optimized with plasmid pNW33N.The result revealed that the highest transformation efficiency of 6.07×104 cfu·μg-1 DNA in strain NCD-2 could be obtained under the optimized condition,the cells of strain NCD-2 electroporated at 14.0 kV·cm-1 and 200 Ω after incubated at 150 r·min-1 and 37 ℃ in growth medium for 3.5 h.

关 键 词：枯草芽孢杆菌 电击转化 转化效率 

分 类 号：S476.8[农业科学—农业昆虫与害虫防治] Q78[农业科学—植物保护]