Functional Analysis Of Cold-temperature Bacteria Promoter

Promoter is an important element for the microbial genetic manipulation, earlier we use psychrotrophs Serratia fonticola strain MY 1402 as the host to establish a basic genetic operating system. Through the construction of Library screening, get seven has low temperature promoter activity fragments.This research focuses on several potential low temperature promoters further analysis and research, we put the recombinant plasmid pUEl, pUE2, pUE3, pUE4, pUE8, pUE9, pUE11 into host cell by electroporation, and detecting the activity of the transformants under the different kanamycin concentration and temperature, the results show that seven promoter fragments have the starting transcriptional activity with room temperature (37?) and low temperature (15?), activity of the strength order p3>p9>p8>p1>p11>p4>p2, but its activity in the two temperatures, there was no significant difference, preliminary indication of the seven promoters is not high sensitivity to temperature.Subsequently, through promoter prediction online website to analyzed their core promoter sequence, and experiment validation the strong activity and fragment size more appropriate of p1, p3 and p8, chemical synthetic theoretical prediction of the core sequence and is connected to the upstream of the reporter gene, by kanamycin screening of promoter activity's recombinants, corresponding to the predicted core promoter fragments are p1-1? p3-1?p3-3?p8-1?p8-4.The recombinant plasmid was transformed into host, using kanamycin concentration gradient, by qRT-PCR was studied quantitatively accurate transcription activity of core promoter fragment at the culture temperature 37? and 15?, the results show that containing the predicted core promoter of recombinants in cultured at 37 ?, p1-1, p3-3 and p8-1 were full promoter activity 72%?85% and 81%. But when the culture temperature is lowered, almost all predictable core promoter loss of low temperature start-up activity, were full promoter activity 37%?41% ? 56%. For further testing of the core sequence of the promoter activity, build the probe plasmid pUG and using GFP as report gene, separately detected the p3, p8 and their core sequence, the results show that only the recombinant plasmid pUG3 with fluorescence, further analyses of the p3. Based on the website predictable core promoter extension respectively 100bp of the upstream and downstream, try to retain the core promoter upstream and downstream transcription initiation components needed, to accurately determine the real core promoter sequence, after testing found that recombinant of p3S1 fragments can produce fluorescence; Then p3S1 is further split, obtained the three promoter fragments p3S1-1? p3S1-2 and p3Sl-3. Microscopic examination showed that the p3S1-1 and p3S1-3 has fluorescence, precise positioning of the core promoter sequence to 129bp, and further identified by an only 20bp key sequence but able to decide p3 promoter transcription initiation.