嗜盐古菌启动子DNA片段的功能检测

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。

Function of promoter DNA fragments from halophilic archaea in Escherichia coli

RM07 and RM13 DNA fragments could function as promoter in Escherichia coli, which were isolated from an archaeon Halobacterium halobium R1. In the present study, promoter activities of these two fragments were confirmed by beta-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. coli DH5alpha. The beta-galactosidase activity of TE132 was higher than that of TE07-2. Both TE07 and TE131 had weak beta-galactosidase activity. Then the heat output of E. coli DH5alpha and its transformants had been detected by a microcalorimetric method at 37 degrees C. Compared with E. coli DH5alpha, the growth rate constant of E. coli 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, beta-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. coli. The higher beta-galactosidase activity of E. coli was, the lower its growth rate constant was. At the meantime, Microcalorimetric studies implied that 700bps of RM13 ( RM131) fragment would have stronger promoter activity than RM13. Microcalorimetry may be used as a new approach for analyzing the regulation of foreign gene expression.