同源重组法构建枯草芽孢杆菌核黄素操纵子突变株  

Construction of Bacillus subtilis Riboflavin Operon Mutant by Homologous Recombination

作　　者：张西锋[1] 郭蔼光[1] 

机构地区：[1]西北农林科技大学生命科学学院/陕西省农业分子生物学重点实验室,陕西杨凌712100

出　　处：《武汉大学学报（理学版）》2009年第3期354-358,共5页Journal of Wuhan University:Natural Science Edition

基　　金：国家重点新产品计划项目(2003ED760039)

摘　　要：为了解除核黄素合成的反馈调节和提高核黄素的产量,以受体菌的核黄素操纵子为同源重组的指导序列,构建了整合表达载体pB1和pB2,通过双交叉同源重组的方法将线性化的pB1和pB2分别整合到受体菌的染色体上.构建了核黄素操纵子的rfn框和启动子ribP1被pB9启动子替换,并在ribB与ribA基因之间引入PnprE启动子的枯草芽孢杆菌核黄素操纵子突变株GJ04和GJ05.通过PCR方法验证了同源重组的正确性.基因工程菌遗传稳定,能分泌产生核黄素.发酵摇瓶实验结果表明:同源重组突变后的菌株GJ04和GJ05的核黄素产量明显高于具有玫瑰黄素抗性标记的菌株GJ02,发酵72 h分别提高了8.6%和11.2%.In order to overcome the inhibition in riboflavin bio-synthesis and enhance riboflavin production, plasmids pB1 and pB2 were constructed by the homologous recombination vectors of the recipient riboflavin operon. Using the method of double crossover recombination between plasmids and chromosomes, plasmids pB1 and pB2 were linearized and integrated into receptors to get two strains named B. subtilis GJ04 and GJ05, with rfn and ribPl promoter being replaced by pB9 promoter and Promoter PnprE being inserted between ribB and ribA gene. The homologous recombination events were confirmed by PCR methods. The obtained strains were genetically stable and could produce riboflavin. The results of shake flask shown that： the production of riboflavin from GJ04 and GJ05 were 8.6% and 11.2% higher in 72 hour than that from the GJ02 with a resistance to roseoflavin.

关 键 词：同源重组 枯草芽孢杆菌 核黄素 操纵子 

分 类 号：Q786[生物学—分子生物学] S852.61[农业科学—基础兽医学]