摘要
用大肠杆菌启动子探测质粒pSDS I (Ap^r,Tc^s)从钝齿棒杆菌(Corynebacterium crenatum)6282染色体的HindⅢ酶切片段中,克隆到两个具有启动功能的DNA片段,分别将两个重组质粒命名为pSDB5和pSDB21。含有这两个质粒的菌株均可以在含300μg/ml Tc的平板上生长。通过酶切分析,pSDB5的插入片段为1.6kb,pSDB21的插入片段为3.4kb,并分别作出了它们的限制性酶切图谱。对pSDB21利用其EcoR Ⅰ和BglⅡ位点,通过亚克隆删除了与启动功能无关片段,构建成pSDB210和pSDB211,从而使启动子定位于约0.1kb的BglⅡ/HindⅢ外源片段上。分子杂交实验证明所得到的这两个具有启动功能的DNA片段确实来源于钝齿棒杆菌6282的染色体DNA。
Two promoter functional fragments have been cloned from the chromosomal DNA of Corynebacterium crenatum 6282 using the promoter-probe vector pSDSI (Apr, Tcr, 5.6kb). Two recombinant plasmids which resumed Tc resistance were named pSDB5 and pSDB21. The restriction maps of them have been determined. The strains haboring them all could grow on the plates containing 300μg/ml Tc. The inserted fragment of pSDB5 was 1. 6kb, and of pSDB21 was 3. 4kb. Two subcloned plasmids of pSDB21 were constructed with the aid of EcoRI and Bgl Ⅱ sites respectively. By removing the non-promoter functional fragments of pSDB21, its promoter of Tcr gene was oriented on the 0. 1kb Bgl Ⅱ/Hind Ⅲ foreign fragment. Southern blot hybridization showed that both of the two promoter functional fragments were from the chromosomal DNA of Corynebacterium crenatum 6282.
出处
《微生物学报》
CAS
CSCD
北大核心
1995年第1期7-13,共7页
Acta Microbiologica Sinica
关键词
启动子
酶谱分析
定位
棒状杆菌属
Corynebacterium, Promoter, Restriction map, Orientation.
