摘要
为了构建能稳定表达外源基因且无抗性标记基因的枯草芽孢杆菌(Bacillus subtilis)工程菌。采用聚合酶链式反应(PCR)技术,分两段扩增定位于B.subtilis BJ3-2染色体上的谷氨酰胺酶基因(glsA)上下游基因片段作为同源臂。将同源臂及卡那霉素基因(Kan)克隆入温敏型载体pKSV7,构建了双交换载体pKUKD。并利用pKUKD质粒将kan基因定点整合入BJ3-2染色体上,通过Kan抗性筛选获得glsA敲除菌株BJ-kan。经检测BJ3-2菌株谷氨酰胺酶活力比重组菌株高3.2倍。结果显示:BJ3-2染色体上的基因可通过同源重组方式进行敲除与置换,为今后BJ3-2菌株以及野生型B.subtilis基因敲除与置换提供了更加便捷的方法。
To efficiently construct resistance gene-free Bacillius subtilis engineered strain which can stably express heterologous gene,using PCR technique,the ends gene of glsA located in chromosome of BJ3-2 strain as homologous arms have been amplified and constructed knocking-out vector pKUKD for dual-exchanging with the Kan gene on the basis of plasmid pKSV7,a temperature sensitive plasmid.A recombinant strain B.subtilis BJ-Kan containing Kan gene was obtained in its chromosome via the dual-exchanging plasmid pKUKD and the kanamycin resistance screening.The detection results showed that glutaminase activity in strain B J3-2 was 3.2 times higher than that in recombinant strain.All the analysis showed that the gene of BJ3-2 could be knocked out and replaced by the way of homologous recombination,and all the results of this experiment provided a convenient recombination system for gene-replacing in B J3-2 or other wild B.subtilis.
出处
《中国酿造》
CAS
2014年第2期28-31,共4页
China Brewing
基金
国家自然科学基金资助项目(31260394)
贵州省重大专项(黔科合重大专项字[2013]6013号)
贵阳市科技计划项目(筑科工合同字[2010]第1-68号)
