同源重组法构建枯草芽孢杆菌dhbC基因缺失突变株和回复株  

Construction of Bacillus subtilis CAS15 dhbC-and dhbC+ Mutants by Using the Homologous Recombination Method

作　　者：余贤美[1,2,3] 林超[1,4] 杨芩[1,4] 李锐[1,2,3] 贺春萍[1,2,3] 郑服丛[1,4] 

机构地区：[1]中国热带农业科学院环境与植物保护研究所,海南儋州571737 [2]农业部热带农林有害生物入侵监测与控制重点开放实验室,海南儋州571737 [3]海南省热带农业有害生物检测监控重点实验室,海南儋州571737 [4]海南大学环境与植物保护学院,海南儋州571737

出　　处：《热带作物学报》2009年第10期1517-1521,共5页Chinese Journal of Tropical Crops

基　　金：中央级公益性科研院所基本科研业务费专项资助项目(No.2008hzs1J013;2008hzs1J014);国家科技支撑计划(No.2007BAD48B04);公益性行业(农业)科研专项(No.nyhyzx07-033-2-3)资助

摘　　要：为了验证dhbC基因的功能,以质粒pEGFP-N1为模板,通过PCR扩增获得新霉素抗性基因(neor)DNA片段,构建重组质粒pMD18-neo,电击转化法将pMD18-neo导入枯草芽孢杆菌Bacillus subtilis CAS15感受态细胞中,使neor基因片段与dhbC基因片段发生置换,获得dhbC基因缺失突变株。再通过电击转化将dhbC基因全长编码序列导入dhbC基因缺失突变株CAS15 dhbC-del,获得dhbC基因回复株CAS15dhbC-com。经CAS平板法检测表明,CAS15 dhbC-del不能产生橘黄色晕圈,而CAS15 dhbC-com产生与CAS一致的橘黄色晕圈,证明了dhbC基因与嗜铁素的合成密切相关。neor DNA fragments were obtained by PCR using plasmid EGFP-Nlas templates, and the recombinant plasmid pMD 18-neo was constructed. The pMD 18-neo was transformed into the competent cell of B. subtilis CAS15 via eleetroporation, and dhbC deletion mutant CAS15 dhbC-del was obtained by replacing the dhbC gene with neor DNA fragments. Then the dhbC complement mutant CAS15 dhbC-com was obtained by retransforming the dhbC gene into CAS15 dhbC-del via electroporation. Finally, CAS15 dhbC-del and CAS15 dhbC-com were inoculated on CAS agar plate. The result showed that CAS15 dhbC-com produced a significant orange halo as CAS15, while CAS15 dhbC-del does not, which indicated that the dhbC gene plays an important role in the siderophore production of CAS15.

关 键 词：枯草芽孢杆菌 同源重组 缺失突变 基因回复 

分 类 号：Q81[生物学—生物工程] Q93