用转座子TnYLB-1构建枯草芽孢杆菌EDR4突变体库

Construction of Mutant Library of Bacillus subtilis EDR4 Using TnYLB-1 Transposon Insertion

为筛选枯草芽孢杆菌EDR4拮抗相关基因,利用转座子TnYLB-1构建该菌株的插入突变体库。通过对种子液OD600值、转接培养液Ⅰ继续培养OD600值及转接培养液Ⅱ后的孵育时间3因素设计正交试验,转化过程中加入pMarA质粒的不同量及质粒与感受态细胞共培养的不同时间研究2因素对转化效率的影响,确定pMarA对EDR4的转化体系。通过50℃高温诱导培养产生EDR4的插入突变体,挑取单菌落2 756个,构建EDR4的插入突变体库,随机挑选14株突变体经PCR检测转座子TnYLB-1序列,发现转座子已成功插入到EDR4基因组中;进一步通过Southern杂交验证,发现转座子TnYLB-1主要以单拷贝形式随机插入,也有部分多拷贝插入。此突变体库的建立可为以后筛选该菌株拮抗相关基因等研究奠定基础。

In order to identify antagonism^-related genes of Bacillus subtilis EDR4,a mutant library was constructed using TnYLB-1transposon insertion.By designing an orthogonal experimental containing three culture times,adding different plasmid amounts and setting different co-cultivation times of plasmid and competence cell,the plasmid pMarA which carries the transposon TnYLB-1was transformed into EDR4 and a transformation system for EDR4 was constructed.The insertion mutant library containing 2 756 mutants was constructed under the condition of 50 ℃ high temperature,PCR amplification of TnYLB-1analyses of 14 mutants showed that the insertion library was reliable.The mutant library laid the foundation for searching the new antagonism^-related genes in EDR4.The result of southern hybridization showed that transposon TnYLB-1had inserted into Bacillus subtilis EDR4 randomly,the mutants were inserted by a single copy,and also some multiple copies appeared.