微量热技术检测古生菌启动子DNA片段的启动功能

Microcalorimetric Studies on the Promoter Function in Escherichia coli HB101 from Halobacterium Halobium Chromosome DNA

用微量热法研究了大肠杆菌HB10 1及其重组菌株的代谢特征 .将不同大小的来源于嗜盐古生菌染色体DNA的启动子片段插入启动子探针载体中的氯霉素抗性基因前 ,获得重组菌株 .结果表明重组菌株的半抑制浓度与DNA片段的启动功能大小是一致的 ,并且质粒的复制几乎不影响菌株的生长及代谢 .培养基中加入氯霉素后 ,启动子启动氯霉素抗性基因表达 ,生长速率 (k)降低 ,最大峰的出现时间 (tp)延长 ,最大峰值 (pm)也随之降低 .在一定范围内 ,抗生素浓度与tp、pm 呈线性关系 .本研究结果直接证明了来自嗜盐古生菌的RM0 7、RM0 8和RM13片段在大肠杆菌中是有启动功能的 ,而且还表明基因表达量的高低与释放的代谢热变化相一致 .

In this paper the heat output of Escherichia coli HB101 and its four different plasmid transformed strains were detected by an LKB2277 heat conduction microcalorimeter. The promoter DNA fragments from archaeal halophiles were inserted upstream chloramphenicol acetyltransferase gene in pKK232-8 plasmid. The results showed that the half-inhibitory concentration was in accordance with the promoter function. When HB101/pKK232-8 was cultured in LB medium, its growth rate was about the same as HB101. It suggested that the replication of plasmids would not affect growth of the receipt strain. While gene expressions were promoted as the antibiotics were added into LB medium, the growth rate (k) and the maximum heat power (p m) were all decreased; the peak time (t p) was prolonged. As the Chloramphenicol(Cm) concentration increased, the growth of E. coli was inhibited. The higher the Cm concentration in LB medium was, the lower the k value was. The results confirmed that RM07, RM08 and RM13 promoter DNA fragments could active as promoters in E. coli, and that gene expression need a lot of energy. Archaea is a special living form. Microcalorimetry may be used as an accurate method to detect archaeal gene expression and regulation, and discover their genetic and metabolic characteristics.