| 摘要: |
| 甘油醛 3 磷酸脱氢酶基因（GAP）作为常见的持家基因在微生物中普遍表达，因而其启动子（PGAP）被认为是一个强的组成型启动子；首先，预测并克隆了双叉双歧杆菌的GAP启动子，并利用葡萄糖醛酸苷酶基因为报告基因，分析了PGAP在大肠杆菌和双歧杆菌中驱动表达的强度；不同碳源的发酵实验表明：PGAP在葡萄糖培养基中强度最高，而3种底物类似物对PGAP强度的影响各不相同;对PGAP预测的 10区进行了系列点突变，具有更接近sigma 70启动子共有序列的突变体pMGAP3表现出更高的驱动强度；最后，通过对代表性双歧杆菌PGAP序列的比对，表明在一些核心区域，如可能的TATA盒、转录起始位点、核糖体结合位点高度保守。 |
| 关键词: GAP 启动子 双歧杆菌 点突变 |
| DOI： |
| 分类号: |
| 基金项目: |
|
| Analysis of A Novel GAP Promoter and Construction of Its Site Directed Mutants |
|
SUN Xue jiao, LI Zhao yang, LI Bing, SUN Zhong ke
|
| 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 using β 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). |
| Key words: GAP promoter bifidobacteria site directed mutation |
