摘要
地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difBs序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difBs序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI′::difGm,通过电击转化法将质粒pHY-XI′::difGm导入B.lichen-iformisATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer/dif位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。
Bacillus licheniformis is one of the most important strains for industrial biotechnological processes. A simple and effective method for gene deletion is fundamental for its genetic improvement. It has been disclosed that Bacillus subtilis difB8 has been successfully applied for its gene disruption in multiple sites. In present studies, B. licheniformis difBLi, a B. subtilis difs-homolog, has been identified in genome of B. licheniformis ATCC14580. Two recombinant plasmids pMDlg-difGm and pHY-XI＇： ： difGm containing the gentamicin resistant cassettes flanked by d/fBLi sites were constructed. The plasmid pHY-XI＇ ： ： difGm was transformed into B. licheniformis ATCC14580. Following regeneration of the transformants, the gcntamicin resistant cassette was efficiently removed via homologous recombination mediated by the native Xer recombinase acting to resolve the two directly repeated dif sites to a single site. It verifies the function of the dif sequence in B. licheniformis and may provide a new tool for the deletion of the multiple genes in B. licheniformis genome.
出处
《微生物学杂志》
CAS
CSCD
2009年第4期11-15,共5页
Journal of Microbiology
基金
国家高技术研究发展计划(863计划
2006AA020204)
