lux基因发光乳球菌构建方法的研究

Studies on the Construction Method of Luminescent Lactococcus

研究了构建lux基因发光乳球菌的方法。研究结果表明,利用设计和合成的低聚核苷酸引物及带有luxCDABE基因簇的质粒pSB377为模板,用PCR技术可以扩增出luxAB和luxCDABE基因片断。选作lux基因的媒介物质粒pMG36e,含有一个在革兰氏阴性、阳性菌中都能启动的复制起始点,与乳球菌启动子P32之下的多克隆位点连接,它能使lux基因成功地插入载体上,并在乳球菌中得以表达。在适宜的培养和电击条件下可以将重组质粒pMG36e-luxAB和pMG36e-luxCDABE转入乳球菌中,使普通的乳球菌变成带lux基因的发光乳球菌。lux基因发光乳球菌的理想的培养基是GMl7而不是M17。在液体培养基中培养时大质粒pMG36e-luxCDABE在无选择压力时其遗传特性不稳定,而小质粒pMG36e-luxAB具有稳定的遗传特性。

The research here is developing construction method of lux-marked luminescent lactococcus. The results showed using oligonucleotide primers designed and synthesized in this work and a plasmid pSB377 containing luxCDABE gene cluster of xenorhabdus luminescens as the template, luxAB and luxCDABE genes were amplified by PCR. The pMG36e, chosen as the vector of lux genes contains a replication origin, which can initiate replication in both gram-negative and gram-positive bacteria. A polyclone site closely downstream the promoter of lactococcrs, p32 facilitate insertion of lux genes in the vector, and expression of lux genes in lactococcus. When cell concentration of lactococcus lactis and electroprating conditions were perfect, the recombinant plasmids, pMG36e-luxAB or pMG36e-luxCDABE could be transformed into the lactococcus lactis by electroperation. GM17 was found a better medium for the luminescent lactococcus lactis than M17. When incubated in the liquid medium, GM17 or M17 without erythromycin, large plasmid (pMG36e-luxCDABE) of the luminescent lactococ-cus lactis would lose soon, and small plasmid (pMG36e-luxAB) can be retained stably.