Abstract:
Promoter probe vector pUE was constructed and used to isolate DNA fragments with promoter activity from aPseudomonas sp.strain MY1402 which can grow at low temperatures. Escherichia coli strain DH5 was used as a host for this study.Seven recombinant plasmids containing seven fragments of different size were obtained.One of the plasmids pUE1 which contained fragment p1 was transformed into MY1402 for promoter activity analysis.The results showed that the highest kanamycin concentration that the positive transformant MY1402/pUE1 could resist was 250 mg/L.Promoter activity of pUE1 at low temperature was also analyzed,similar growth rate of MY1402/pUE1 at 15 ℃ to that at 28 ℃ was observed,which indicated that fragment p1 had promoter activity at low temperature.Predicted core sequence p1a of the fragment p1 was synthesized and subcloned into pUE,generating recombinant pUE1a.Using the same methods,promoter activity of pUE1a was subjected to analyze.The results showed that the promoter activity of the predicted core sequence p1a was almost as strong as that of p1 at 28 ℃ but decreased significantly at 15 ℃,no obvious growth of MY1402 transformed with pUE1a was observed in the presence of kanamycin concentrations over 100 mg/L at 15 ℃,indicating the flanking sequences were involved with the promoter activity of p1 at low temperature.