嗜盐古菌启动子DNA片段的功能检测  

Function of promoter DNA fragments from halophilic archaea in Escherichia coli

作　　者：黄玉屏[1] 刘鹏[2] 刘义[2] 沈韫芬[3] 沈萍[1] 

机构地区：[1]武汉大学生命科学学院 [2]武汉大学化学与分子科学学院,武汉430072 [3]中国科学院水生生物研究所,武汉430072

出　　处：《微生物学报》2006年第2期200-204,共5页Acta Microbiologica Sinica

基　　金：科技部973专项基金(2004CB719603);国家自然科学基金(30470033);中国博士后科学基金(2003034506)~~

摘　　要：将来源于嗜盐古菌染色体DNA的启动子片段RM07或RM13插入到启动子探针载体pYLZ_2的报告基因lacZ之前,通过β_半乳糖苷酶酶活性的检测,进一步确证RM07和RM13片段在大肠杆菌(Escherichia coli)中的启动功能。同时用微量热技术检测了大肠杆菌DH5α及其重组菌株在LB培养基中37℃生长过程的热输出功率。T2(pYLZ_2)、TE07(pYL726)、TE07_2(pYL702)、TE131(pYL131)和TE132(pYL132)菌株的生长速率分别比大肠杆菌DH5α降低了6.5%、11%4、1.1%4、7.5%和42.7%。当启动子启动了基因表达时,菌株的生长速率显著降低,热力学参数与酶活性检测结果有较好的一致性。微量热结果表明基因的表达比质粒DNA的复制过程需要消耗更多的能量,对细菌的生理代谢有较大改变。微量热技术为检测基因的表达和转录调控提供了新的方法和思路。RM07 and RM13 DNA fragments could function as promoter in Escherichia coil, which were isolated from an archaeon Halobacterium halobium RI. In the present study, promoter activities of these two fragments were confirmed by β-galactosidase activity analysis and microcalorimetric studies. They were cloned into promoter-probe vector pYLZ-2 respectively. Four recombinant strains TE07, TE07-2, TE131 and TE132 were obtained, and all fragments were found to be active in E. coil DH5α. The β-galactosidase activity of TE132 was higher than that of TE07-2. Both TE07 and TE131 had weak β-galactosidase activity. Then the heat output of E. coil DH5α and its transformants had been detected by a microcalorimetric method at 37 ℃. Compared with E. coil DH5α, the growth rate constant of E. coil T2（pYLZ-2）, TE07, TE07-2, TE131 and TE132 strain was reduced 6.5%, 11% ,41.1%, 47.5% and 42.7% respectively. When IPTG was added to LB medium, β-galactosidase activity and heat output had been enhanced slightly in all strains. The results suggested that there was close correspondence between promoter activity and microcalorimetric results, and the heat output of growth was mainly affected by gene expression in E. coll. The higher β-galactosidase activity of E. coil was, the lower its growth rate constant was. At the meantime, Microcalorimetric studies imptied that 700bps of RM13 （RM13- 1） fragment would have stronger promoter activity than RM13. Microcalorimetry may be used as a new approach for analyzing the regulation of foreign gene expression.

关 键 词：启动子 微量热技术 Β-半乳糖苷酶基因 嗜盐古菌 

分 类 号：Q933[生物学—微生物学]