【作者】 朱建裕；

【导师】 沈萍； 胡岳华；

【作者基本信息】 中南大学 ， 微生物学， 2005， 博士

【摘要】 利用大肠杆菌启动子探针载体pKK232-8,从极端嗜盐古生菌中基因组DNA分离得到一个能在真细菌域(大肠杆菌)中具有启动子活性的RM10 DNA片段。DNA序列查询发现它包含了嗜盐古生菌rad25基因部分编码序列和上游调控序列;DNA序列分析表明该片段既含有类似嗜盐古生菌启动子和真核生物RNA聚合酶Ⅱ启动子的TATA box特征序列等,也含有典型的真细菌启动子-35和-10区特征序列。 在嗜盐古生菌中,以β-半乳糖苷酶基因(bgaH)为报告基因,构建了RM10片段和报告基因相融合的重组质粒,通过检测报告基因转录的mRNA和表达的酶活性,证明了RM10 DNA片段的确具有启动子功能。通过构建了一系列RM10不同缺失片段融合到报告基因上游的重组质粒,检测了RM10不同缺失片段的启动子活性大小,在嗜盐古生菌中浓缩定位了RM10片段中对于启动子活性有影响的重要功能区,结果表明:rad25基因5’端上游含有TATA box和BRE特征序列(-305至-130)是片段中对启动子活性有影响的关键功能区。 在大肠杆菌中,利用氯霉素抗性基因(cat)作为报告基因,构建了RM10片段和报告基因的融合质粒,通过检测报告基因的转录和抗性水平,结果表明RM10片段在大肠杆菌具有启动子功能。通过构建缺失片段插入报告基因上游的重组质粒,在大肠杆菌对RM10片段进行了缺失突变分析,确定了rad25基因5’端上游序列包含TATAⅠ特征序列的-305～-250区段是影响RM10片段在大肠杆菌的启动子活性关键部分。结果说明rad25基因5’端上游序列在大肠杆菌有启动子功能和在嗜盐古菌中有启动子功能在序列上既存在差异,同时又是相互联系的。 在酿酒酵母菌和哺乳动物细胞中,分别利用G418抗性基因(neo~r)、绿色荧光蛋白基因(egfp)、荧光素酶基因(Luc+)为报告基因,构建各种RM10片段和报告基因相融合的重组质粒,通过检测报告基因表达的抗性水平、绿色荧光和酶活性,证实RM10在真核生物中具有启动子功能。通过构建缺失片段融合到报告基因上游的重组质粒,对RM10片段进行了缺失突变分析,结果表明:在酿酒酵母中,含有TATAⅣ和多个GGCGG box特征序列的-1767～-889区段对RM10片段在酵母菌中具有强启动子活性很重要,包含TATAⅢ特征序列的-889～-403区段是RM10片段在酵母菌中有启动子活性的基本的区段,包含TATAⅡ特征序列的-403～-329区段对RM10片段在酵母菌中有启动子活性有一定影响。在动物更多还原

【Abstract】 In this dissertation, DNA fragment designated RM10 was isolated from the chromosomal DNA of the halophilic archaea Halobacterium halobium by using the Escherichia coli promoter-probe vector pKK232-8 and was shown to confer promoter activity in Escherichia coli. sequence searches for homology in Genbank of NCBI revealed that RM10 contained the sequence of 5’upstream region of rad25 gene in Halobacterium salinarium. Sequence analysis revealed that RM10 fragment contained characteristic sequences similar to eukaryal and archaeal promoter-TATA box as well as the typical -35 and -10 box consensus sequences of bacterial promoter.The RM10-bgaH gene fusion plasmids were constructed and transformed into halophilic archaea Haloferax volcanii using the bgaH gene as the reporter genes, Through determining the enzymatic activity and RT-PCR analysis, it was confirmed that RM10 fragments conferred promoter activity in Haloferax volcanii. Deletion analysis of RM10 was performed in Haloferax volcanii, Through detecting the promoter activity of various deletion fragments of RM10, the important functional regions within RM10 which could influence the promoter activity were identified. The results revealed that the -305 to -130bp region of 5’upstream region of rad25 gene containing the typical TATA box and BRE sequences of archaeal promoter was the key region responsible for the basal promoter activity of RM10 in Haloferax volcanii.Using the cat gene as the reporter genes, the RM10-reporter gene fusion plasmid was constructed and transformed into Escherichia coli. Through detecting the antibiotic resistance level and RT-PCR analysis, it was confirmed that RM10 conferred promoter activity in Escherichia coli. Through deleting analysis of RM07 in Escherichia coli ,the important functional regions within RM10 which could influence the promoter activity were identified. The results revealed that the -305 to -250 region of 5’upstream region of rad25 containing the TATA Ⅰ sequences was the important functional region responsible for the basal promoter activity of RM10 in Escherichia coli. The promoter functional region of Bacteria and archaeal were related.Using the neo,egfp and luc+ gene as the reporter genes, various RM10-reportergene fusion plasmids were constructed and transformed or transfeced into Saccharomyces cerevisiae and superior mammalian cell respectively. Through detecting the antibiotic resistance level, detecting EGFP fluorescence expression and determining the enzymatic activity, it was confirmed that RMIO conferred promoter activity in Saccharomyces cerevisiae and superior mammalian cell. Deletion analysis of RMIO was performed in Saccharomyces cerevisiae and superior mammalian cell. The results revealed that the -1767 to -889 region of 5’upstream region of rad25 gene containing characteristic sequences of TATA IV and GGCGG box was the functional region responsible for the strong promoter activity ,and the -889 to -403 region of 5’upstream region of rad25 gene containing characteristic sequences of TATAIII was the functional region responsible for the basal promoter activity of RMIO in Saccharomyces cerevisiae. In superior mammalian cell, the -889 to +1 region of 5’upstream region of rad25 gene containing characteristic sequences of TATAIII, TATA II and TATA I was the functional region responsible for the basal promoter activity and these functional region were related in Saccharomyces cerevisiae and superior mammalian cell.An efficient, quantitative, inexpensive and versatile microcalorimetry was successfully applied in studying the structure and function of promoter in Saccharomyces cerevisiae and Escherichia coli.更多还原