嗜热脂肪芽孢杆菌pgiB基因上游452bp序列的功能分析

Analysis of Function of the 452bp Sequence Proceeding the pgiB Gene in Bacillus stearothermophilus

用化学诱变剂N 甲基 N′ 硝基N 亚硝基胍进行随机诱变 ,获得了穿梭启动子探测质粒pPGV5的温度抗性突变型pPGV5 (tr6 5 ) ,序列分析发现质粒上卡那霉素核苷转移酶基因kan的 +2 3 8位碱基发生了G→T的单点突变。以来自嗜热脂肪芽孢杆菌FDTP 3菌株的耐热邻苯二酚 2 ,3双加氧酶基因pheB作为报道基因 ,构建了转录融合质粒pPGVPB45 2 ,用高压电穿孔法将其转化嗜热脂肪芽孢杆菌 ,通过报道蛋白活性的分析 ,证明了嗜热脂肪芽孢杆菌T5 2 1菌株的 6 磷酸葡萄糖异构酶同工酶基因pgiB上游含启动子样序列的 42

By random mutagenesis with chemical mutagen N-methyl-N′-nitro-N-nitrosoguanidine,a shuttle promoter probe vetor mutant pPGV5 (tr65) was obtained.A single site mutation from G to T was discovered at nucleotide+238 of kan gene encoding kanamycin nucleotidyltransferae by sequence analysis.Using the gene pheB that encodes thermostable catechol 2′3-dioxygenase fromBacillus stearothermophilus FDTP-3 as the reporter gene,a transcriptional fusion plasmid pPGVPB452 was constructed and electroportated into B.stearothermophilus.By analyzing the reporter protein,it proved that the 452bp sequence proceeding the coding region of phosphoglucoisomerase pgiB gene from B.stearothermophilus T521,which contained a putative promoter by sequence analysis,but has no transcriptional activity in B.stearothermophilus.