侧孢短芽孢杆菌X10启动子活性片段的克隆和序列分析

Cloning and Sequence Analysis of Fragments with Promoter Activity in Brevibacillus laterosporus X10

提取了侧孢短芽孢杆菌X10的基因组DNA,以绿色荧光蛋白基因（green fluorescent protein,gfp）为报告基因,以启动子探针pUC19-GFP为载体,通过鸟枪法在大肠杆菌DH5α中构建了X10的启动子文库,通过筛选获得了14个阳性克隆,编号为P1~P14。测定了阳性克隆子的荧光强度,结果表明P6中gfp基因的启动子活性最强,它的荧光强度达到了355.67,而P14中gfp基因的启动子活性最弱,它的荧光强度只有211.67。对P6克隆中的重组质粒的插入片段进行了测序和序列分析。

High quality genomic DNA of Brevibacillus laterosporus XIO was extracted. The promoter library of X10 was constructed in Escherichia coli DHSct with pUC19-GFP as promoter vector by shotgun-cloning method. 14 positive clones designed as P1 - P14 were obtained by screening, the fluorescent intensity of which was assayed to compare the promoting strength of the promoters. The resuits showed that the promoter of gfp in P6 was the strongest with fluorescent intensity of 355.67 ,while the promoter of gfp in P14 was the weakest with fluorescent intensity of 211.67. The inserting fragments in the recombinant from P6 were sequenced and analyzed.