摘要
枯草芽胞杆菌9407(B9407)是1株对苹果轮纹病具有显著防效的生防菌株。为深入研究该菌株防病的分子机制,利用携带转座子TnYLB-1的温敏型质粒pMarA构建了B9407的突变体库。通过电击转化将pMarA转化至B9407,经过30℃培养,37℃转座诱变,50℃进行pMarA质粒消除,最后通过抗性筛选,获得3 500个转座子插入突变的单克隆,构建了B9407的突变体库。该转座子TnYLB-1的转座效率达到2.4×10-3,pMarA的质粒消除率达97.3%。随机挑选12株突变子进行PCR及Southern blotting杂交,结果表明:转座子TnYLB-1能够随机插入到B9407基因组上,大于90%的突变子为转座子单拷贝插入。B9407TnYLB-1转座子突变体库的构建为筛选和研究影响该菌株生防功能相关的基因并揭示其生防机制奠定了基础。
Bacillus subtilis 9407 （B9407） is a beneficial bacteria against apple ring rot disease. In order toexplore the molecular mechanism of B9407, a mutant library was constructed using a temperature sensitive plasmid pMarA, a mariner-based transposon TnYLB-1. The plasmid pMarA was transformed into B9407 at 30 ℃, and induce to transpose at 37℃, followed by kicking out the remained pMarA and screening transponsants on LB plates with different antibiotics. Finally, 3 500 transposants were select ed. The tansposase efficiency of the transposon TnYLB-1 reaches to 2.4× 10^-3 and elimination rate of pMarA plasmid is 97.3%. Southern blotting hybridization analyses of 12 transposants and sequence analyses of the insertion sites are consistent with random transposition and at least 90% of the transposants contain a single transposon insertion site. The construction of B9407 transposon mutant library will provide us a valid tool to identify new genes involved in biological control and understand the biological control mechanisms.
出处
《中国科技论文》
CAS
北大核心
2016年第18期2082-2086,共5页
China Sciencepaper
基金
高等学校博士学科点专项科研基金资助项目(20110008120013)
