多粘类芽胞杆菌SC2基因敲除体系的建立

Construction of gene knock-out system for Paenibacillus polymyxa SC2

摘要:

摘要:【目的】建立多粘类芽胞杆菌SC2 的基因敲除体系。【方法】利用电转化把温敏型自杀质粒pRN5101导入到多粘类芽胞杆菌SC2中。采用基因重组技术敲除SC2 中的多粘菌素基因E(pmxE)，得到突变株SC2-E。利用抗细菌性能检测和高效液相色谱分析合成多粘菌素的能力，来证实pmxE基因是否被敲除。【结果】成功构建了多粘类芽胞杆菌SC2 的基因敲除体系。pRN5101转入SC2后能够在28℃复制，39℃自杀。突变株失去了合成多粘菌素的能力，成功敲除pmxE基因，验证了此体系的可用性。【结论】首次构建了多粘类芽胞杆菌的基因敲除体系，拓展了pRN5101的使用范围，为研究多粘类芽胞杆菌的基因功能提供了高效的遗传操作工具。

Abstract:

Abstract:［Objective］To construct an efficient gene knock-out system for Paenibacillus polymyxa SC2.［Methods］Temperature sensitive plasmid pRN5101 was transformed into P．polymyxa SC2 by electrotransformation．A mutant SC2-E was obtained，in which pmxE was disrupted by homologous recombination． To confirm whether pmxE was knocked out，we used antibacterial activity assay and high performance liquid chromatography to analyze the ability of mutants synthesizing polymyxin.［Results］We developed an efficient gene knock-out system for P. polymyxa SC2. Plasmid of pRN5101 could replicate at 28℃ and suicide at 39℃ in SC2. Mutants lost the ability of synthesizing polymyxin，indicating that pmxE gene was successfully knocked out.［Conclusion］The constructed gene knock-out system for P. polymyxa provides a highefficiency tool to detect genes function for P. polymyxa．