钝齿棒杆菌乳酸脱氢酶基因敲除载体构建

Construction of Corynebacterium crenatum ldhA gene knock-out vector

乳酸脱氢酶催化丙酮酸生成乳酸是钝齿棒杆菌厌氧发酵产乳酸的关键酶。文章以构建乳酸脱氢酶基因敲除载体为目的,通过在乳酸脱氢酶基因片段中的EcoRV酶切位点插入卡那霉素抗性基因的方法构建钝齿棒杆菌乳酸脱氢酶基因敲除载体。结果显示,成功扩增并克隆到乳酸脱氢酶基因与卡那霉素抗性基因,质粒聚合酶链式反应(polymerase chain reaction,PCR)与酶切结果显示,卡那霉素抗性基因准确插入到乳酸脱氢酶基因中的EcoRV酶切位点,钝齿棒杆菌乳酸脱氢酶基因敲除载体构建成功。

When producing lactic acid by Corynebacterium crenatum under anaerobic condition ,the lac‐tate dehydrogenase is the key enzyme which can catalyze the formation of lactic acid from pyruvate .In this paper ,the ldhA gene knock‐out vector was constructed by inserting a km gene into the EcoRV re‐striction site of ldhA .The results indicated that the ldhA and km genes were cloned successfully ,the positive recombinant plasmid was identified by polymerase chain reaction（PCR） and enzyme digestion and the results certified that the km gene was accurately inserted into the EcoRV restriction site of ldhA and the ldhA gene knock‐out vector was successfully constructed .