玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01突变体文库的构建

摘要

To construct a mutant library for zearalenone-degrading bacterium Bacillus amyloliquefaciens MQ01 for cloning zearalenone degrading enzyme gene and defining the molecular mechanisms of zearalenone degradation by MQ01.The shuttle vector pMarA carrying transposon TnYLB-1 was electroporatred into zearalenone-degrading bacterium B.amyloliquefaciens MQ01.The transposon TnYLB-1 was then inserted randomly into the genomic DNA of MQ01 at 50℃.A total of 3 000 positive clones of TnYLB-1 transposon inserted mutation were obtained, and a mutant library for MQ01 was constructed there by.PCR and Southern hybridization revealed that the TnYLB-1 transposon was inserted randomly into the genomic DNA of B.amyloliquefaciens MQ01 in the form of single copy, enabling the screening of transposon mutants which lose the zearalenone-degrading ability from the mutant library, and the cloning of zearalenone-degrading enzyme gene.%构建玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01突变体文库,以期获得丢失玉米赤霉烯酮降解功能的突变子,克隆玉米赤霉烯酮降解酶基因,阐明MQ01降解玉米赤霉烯酮的分子机制.将携带转座子TnYLB-1的穿梭载体pMarA电转化至玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01中,50 ℃ 高温条件下,把穿梭载体pMarA上转座子TnYLB-1随机插入到菌株MQ01基因组中,获得转座子插入突变的阳性克隆,构建MQ01菌株的突变体文库,随机挑选突变子采用PCR和Southern杂交方法进行验证.本研究成功获得了3 000多个TnYLB-1转座子插入突变的阳性克隆,构建了B.amyloliquefaciens MQ01的突变子文库,结果显示TnYLB-1转座子以单拷贝的形式随机插入到B.amyloliquefaciens MQ01的基因组DNA 中,从而可以从转座子突变文库中筛选丢失玉米赤霉烯酮降解功能的转座突变子,从菌株MQ01中克隆玉米赤霉烯酮降解酶基因.