乳酸乳球菌乳脂亚种MG1363中盐诱导启动子的克隆

Cloning of promoters from Lactococcus lactis subsp.cremoris MG1363

为了从乳酸菌中筛选和克隆启动子,实验利用缺失T7启动子的质粒载体PRSET/LacZ直接在大肠杆菌(E.coli)DH5α中分离乳酸乳球菌乳脂亚种(Lactococcus lactis subsp.cremoris)MG1363的基因启动子片段,获得了10多个具有抗氨苄和盐诱导出蓝斑的重组子。反复筛选并对其中一个抗性最高的重组子PRSET-osm进行序列测定和同源性分析发现,所克隆的基因启动子片段来自乳酸乳球菌乳脂亚种MG1363的基因组,并具有原核启动子的保守序列(Pribnow框和Sextama框)。对启动子osm进行进一步序列分析和鉴定发现,其在大肠杆菌BL21中启动LacZ基因的表达,确定osm为盐诱导启动子。

For the purpose of screening and cloning promoters from Lactococcus lactis cremoris MG1363,DNA fragments were separated by using the T7 promoter-less vector PRSET/LacZ in E.coli DH5a. More than 10 fragments were obtained,which were identified based on their ability to confer resistance against Ampicillin and induced by high press. After sequencing and homeology blast,a rec0n was found（PRSET-gro）. It was from Lactococcus/actis cremoris MG1363 and has the identical consensus region Pribnow box and Sextama box. After sequences analysis and identification of osm putative promoter,we found that it could endow expression of LacZ in E. coil BL21 ,moreover,the osm promoter was intended to be osmotic pressure inducible promoter.