短小芽孢杆菌(Bacillus pumilus)DX01转座突变株的构建及转化体系的优化  

Generation of Bacillus pumilus DX01 Mutations with Tn5 Transposon and Optimization of Its Electrotransformation System

作　　者：胡晓璐[1] 沈新迁[1] 傅科鹤[1] 顾振芳[1] 陈云鹏[1] 

机构地区：[1]上海交通大学农业与生物学院,上海200240

出　　处：《上海交通大学学报（农业科学版）》2011年第1期68-74,共7页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家自然科学基金资助课题(31071721);上海市白玉兰科技人才基金(2010B046);农业部都市农业(南方)重点实验室开放课题(09UA005)

摘　　要：Tn5转座子及其衍生载体被广泛应用于革兰氏阴性菌的基因功能研究,但尚未见有利用Tn5转座子导入革兰氏阳性菌短小芽孢杆菌(Bacillus pumilus)DX01菌株的报道。本研究通过构建Tn5转座载体,对短小芽孢杆菌DX01菌株进行了Tn5转座插入诱变,获得了大量的突变株。同时,考察了感受态细胞培养浓度、电击电压、电击缓冲液及复苏培养基的选择等对DX01电转化效果的影响,建立了实验条件下的最佳电转条件。从1 109个转化子中随机抽取20个继代培养15代后,进行了稳定性分析;对转化子的PCR及Southern blot分析证实Tn5已随机插入到DX01基因组中。Tn5 transposon and its derivatives have been widely used to understand gene functions in Gram-negative bacteria.So far,it has no report that Tn5 is able to transform into Gram-positive bacterium Bacillus pumilus DX01.Here,we generated a Tn5 transposon derivative and successfully transformed it into Bacillus pumilus DX01.1 109 transformants displayed resistance to hygromycin B.To further improve the efficience of Tn5 transformation system in B.pumilus,several electrotransfection parameters including cell incubation time,pulsed electric field,electrotransformation buffer and resuscitative buffer,were optimized.Twenty mutations from 1 109 transformants were randomly selected for successive subculture for 15 generations to test their stability.PCR and Southern blotting confirmed that Tn5 transopon integrats into DX01 genome.

关 键 词：短小芽孢杆菌 突变 Tn5转座子 电击转化 

分 类 号：S511[农业科学—作物学] Q343.1[生物学—遗传学]