重庆工商大学学报（自然科学版）2016，Vol.33Issue(6)：117-123,7.DOI:10.16055/j.issn.1672-058X.2016.0006.023
一个GAP启动子的分析及点突变体构建∗
Analysis of A Novel GAP P romoter and Construction of Its Site Directed Mutants
摘要
Abstract
As a house⁃keeping gene, glyceraldehyde 3⁃phosphate dehydrogenase ( GAP ) is ubiquitously expressed in various microorganisms. Therefore, its promoter is believed a strong constitutive promoter. In this study, a novel PGAP was predicted and cloned from Bifidobacterium bifidum. The strength of this promoter in both Escherichia coli and bifidobactteria was probed by enzyme activity assay usingβ⁃glucuronidase ( gusA) as reporter. Furthermore, impacts of different carbon sources and derivatives of GAP substrate on the strength of promoter in bifidobacteria were tested. Sugar fermentation experiments show the promoter is strongest when glucose was used as carbon source, and supplementation of three kinds of substrates influences the strength as well. Site directed mutagenesis ( SDM) of predicted⁃10 region yielded three derivatives with different strengths. The mutant 3, namely pMGAP3 shows the strongest activity, which has more close structure to the consensus sequence of sigma 70 like promoters. In the end, alignment of all representative PGAP sequences in bifidobacteria demonstrates they are very conservative in some core regions, like TATA box, transcription start site ( TSS ) , and ribosome binding site ( RBS) .关键词
GAP/启动子/双歧杆菌/点突变Key words
GAP/promoter/bifidobacteria/site directed mutation分类
生物科学引用本文复制引用
孙雪娇,李昭洋,李兵,孙忠科..一个GAP启动子的分析及点突变体构建∗[J].重庆工商大学学报（自然科学版）,2016,33(6):117-123,7.基金项目
周口师范学院2015年食品微生物科研平台专项基金（ ZKNU2015007）． （）
