【作者】 杨洋；

【导师】 沈萍；

【作者基本信息】 武汉大学 ， 遗传学， 2003， 博士

【摘要】 利用大肠杆菌启动子探针载体pKK232-8，从极端嗜盐古生菌的基因组DNA中分离得到一个在大肠杆菌中具有启动子活性的RM07 DNA片段，DNA序列分析表明它既含有典型的真细菌启动子-35区和-10区保守序列，又具有嗜盐古生菌启动子特征序列DPE(distal promoter element)。 在嗜盐古生菌、大肠杆菌和酿酒酵母中分别以β-半乳糖苷酶基因(bgaH)、二氢叶酸还原酶基因(dhfr)、氯霉素抗性基因(cat)、G418抗性基因(neo)为报告基因，通过构建各种RM07片段和报告基因相融合的重组质粒、酶活测定、抗性水平检测以及逆转录PCR(RT-PCR)检测报告基因转录等方法，研究了RM07 DNA片段的启动子功能，结果证明RM07片段在三域模式生物——古生菌(嗜盐古生菌)、真细菌(大肠杆菌)和真核生物(酵母菌)中均具有启动子活性，能够驱动不同报告基因的转录和表达。 在嗜盐古生菌、大肠杆菌和酵母菌中分别对RM07片段进行了启动子缺失突变分析，构建了一系列RM07不同缺失片段插入报告基因上游的重组质粒，检测了RM07不同缺失片段的启动子活性大小，在三域模式生物中分别浓缩定位了RM07片段中对于启动子活性有影响的重要功能区。结果表明：含有-35区和-10区特征序列的40bp区段是RM07片段在大肠杆菌中具有基础启动子活性的重要功能区；嗜盐古生菌启动子特征序列DPE对于RM07片段在嗜盐古生菌和酵母菌中的启动子活性都是必需的功能元件；RM07片段中的-35区、-10区特征序列和嗜盐古生菌启动子特征序列DPE在启动子功能上具有一定的相关性。 在大肠杆菌中对RM07片段进行了定点诱变分析，结果表明不同碱基突变对RM07片段的启动子活性产生不同的影响，从而进一步精确定位了R材07片段中对在大肠杆菌中的启动子功能有重要作用的关键碱基，初步鉴定了可能的转录起始位点，并且通过改造RM07片段的碱基组成成份大幅提高了其在大肠杆菌中的启动子活性。 本研究还成功地将热化学研究的重要方法—微量量热技术应用于启动子的结构与功能研究，进一步证实了RM07片段在大肠杆菌中的启动子功能，获得了不同的碱基突变影响RM07片段在大肠杆菌中的启动子活性的精确数据，热化学研究结果与生物学的研究结果基本一致。本研究也为启动子功能的研究提供了一种新的更加灵敏便捷的化学与生物学相结合的方法。更多还原

【Abstract】 Using the Escherichia coli promoter probe vector pKK232-8, a 492 bp DNA fragment, designated RM07, was isolated from the chromosomal DNA of the halophilic Archaea, Halobacterium halobium, and was shown to confer promoter activity in Escherichia coli. Sequence analysis revealed that RM07 fragment contained the typical -35 and -10 box consensus sequences of bacterial promoter as well as the characteristic sequence of archaeal promoter-DPE(distal promoter element).Using the bgaH, dhfr, cat and neo gene as the reporter genes, various RM07-reporter gene fusion plasmids were constructed and transformed into Haloferax volcanii, Escherichia coli and Saccharomyces cerevisiae respectively. Through determining the enzymatic activity, detecting the antibiotic resistance level and RT-PCR analysis, it was confirmed that RM07 conferred promoter activity in all three domains of life: Archaea (Haloferax volcanii), Bacteria (Escherichia coli) and Eukarya (Saccharomyces cerevisiae).Deletion analysis of RM07 was performed in Haloferax volcanii, Escherichia coli and Saccharomyces cerevisiae. The recombinant plasmids with different deletion fragments of RM07 inserted upstream of reporter gene were constructed. Through detecting the promoter activity of various deletion fragments of RM07, the important functional regions within RM07 which could influence the promoter activity were identified. The results revealed that the 40bp region containing the typical -35 and -10 box sequences of bacterial promoters was the important functional region responsible for the basal promoter activity of RM07 in Escherichia coli. The characteristic sequence of archaeal promoter-DPE(distal promoter element) was the functional element necessary for thepromoter activity of RM07 in both Archaea (Haloferax volcanif) and Eukarya(Saccharomyces cerevisiae). The promoter functional elements (-35 , -10 box andarchaeal DPE) were related.Site-directed mutagenesis of RM07 was performed in Escherichia coll.Different nucleotide mutations had different effect on the promoter activity of RM07. The critical nucleotide responsible for the promoter function of RM07 in Escherichia colt was determined precisely. The possible transcription initial nucleotide site was also identified. The promoter activity of RM07 in Escherichia coli was improved greatly by modifying the nucleotide component in RM07.In this study, the important thermochemistry research method microcalorimetry was successfully applied in studying the structure and function of promoter in Escherichia coli. The research results of microcalorimetry further confirmed that RM07 could confer promoter activity in Escherichia coli. The different effects of various nucleotide mutations on the promoter activity of RM07 were detected by means of microcalorimetric method. The results were consistent with the results gotten by the traditional biological method. Our work also provided a more sensitive and easily-performed novel method combining the chemical and biological technique for studying the promoter function.更多还原