摘要
以黑暗链霉菌Tt-49基因组为模板,利用PCR方法,扩增安普霉素生物合成关键基因aprH^M的上、下游序列,作为同源交换臂,并以温敏复制型质粒pKC1139为基础,构建用于阻断黑暗链霉菌Tt-49中安普霉素生物合成的重组质粒pHM106。质粒经接合转移进入黑暗链霉菌Tt-49,并筛选得到发生同源双交换工程菌,命名为黑暗链霉菌HM106。通过PCR鉴定,证明工程菌HM106中的aprH^M被tetr替换。对工程菌HM106进行发酵产物分析,结果是其发酵效价下降明显,仅为出发菌株的40%左右。采用薄层层析(TLC)对其组分分析,其安普霉素组分消失,因此,初步判断已成功阻断安普霉素的生物合成。
Two fragments （JHB 1 and JHB2）, which are respectively located upstream and downstream of april-M, were amplified by PCR from S. tenebrarius Tt-49 genomic DNA and were used as exchange arms. The recombinant plasmid priM106, for homologous recombination was constructed from thermal sensitive plasmid pKCl139. The plasmid priM106 was transformed into S. tenebrarius Tt-49 by conjugation, and the homologous double-exchange engineering bacteria was tilted,named S. tenebrarius HM106. It was verified by PCR that the gene aprH-M in the HM106 was replaced by gene tetr. The results of fermentation product analysis of HM106 showed that its fermentation titer decreased obviously, only about 40% of the original strain. It was verified by TLC that the apramycin wes disappeared. So it was speculated that the biosynthesis of apramycin was blocked.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2012年第9期666-670,共5页
Chinese Journal of Antibiotics
基金
国家自然科学基金(项目编号31070093/30801449)
