[Construction of gene knock-out system for Paenibacillus polymyxa SC2].
[Construction of gene knock-out system for Paenibacillus polymyxa SC2].
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发表时间：
2013-12
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通讯作者：
Wendi Zhang;Yanqin Ding;Liangtong Yao;Kai Liu;B. Du
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作者：
Wendi Zhang;Yanqin Ding;Liangtong Yao;Kai Liu;B. Du
目的构建高效的多粘类芽孢杆菌SC2基因敲除体系。方法采用电转化法将温敏质粒pRN5101转化为多黏菌SC2。获得突变体SC2-E，其中pmxE被同源重组破坏。为了确认pmxE是否被敲除，我们采用抗菌活性测定和高效液相色谱法分析突变体合成多粘菌素的能力。结果建立了一个高效的多粘菌SC2基因敲除系统。pRN5101质粒在SC2中可在28℃复制，在39℃自杀。突变体失去了合成多粘菌素的能力，表明pmxE基因被成功敲除。结论构建的多粘菌基因敲除系统为多粘菌基因功能检测提供了一种高效的工具。
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 degrees C and suicide at 39 degrees C 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 high-efficiency tool to detect genes function for P. polymyxa.