肠膜明串珠菌基因失活体系的建立

Construction of Gene Inactivation System for Leuconostoc mesenteroides

构建肠膜明串珠菌的基因失活体系。四环素抗性基因表达盒连接到pTA2 T载体上,在T载体的左右多克隆位点上分别定向插入ack(乙酸激酶)的上、下游同源臂,构建成自杀性同源重组载体,转化明串珠菌,使染色体的ack失活,得到突变型。检测突变型产乙酸的量并利用PCR验证ack失活。与原始菌株相比,突变型的乙酸产量减少49.1%;突变型的ack PCR扩增产物比原始菌株的大1200 bp左右。成功地使ack基因失活,说明肠膜明串珠菌基因失活体系的构建成功。

To develop an efficient gene inactivation system for L. mesenteroides, tetracycline-resistant gene cassettes were ligated with pTA2 T vector. Then upsteam and downsteam flanking regions of the ack (acetate kinase) gene were directionally inserted into left and right MCS (multiple cloning site) of the T vector, respectively. The resulting homologous recombination suicide vector was trans-formed into Leuconostoc. The ack gene of chromosome was inactivated and a mutant strain was obtained. To confirm whether ack was inactivated, the ability of mutant strain synthesizing acetic acid was analyzed and an ack fragment was amplified by PCR. PCR analysis showed that a fragment of ack gene in the mutant strain was 1200 bp longer than that in the original strain. Compared with the original strain, the yield of acetic acid in the mutant was decreased by 49.1%. The results indicated that ack gene was successfully knocked out. The gene inactivation system for L. mesenteroides was constructed.