Cloning and functional analysis of the promoter of a UDP-glycosyltransferase gene from Panax quinquefolium L.

Abstract

In order to explore the function of the Pq3-O-UGT2 promoter, chromosome walking technology was used to isolate the 1399 bp sequence upstream of the ATG initiation codon of Pq3-O-UGT2 from Panax quinquefolium L. Bioinformatics analysis shows that the nucleic acid sequence contains a large number of typical structures unique to eukaryotic promoters and many other important cis-acting regulatory elements, including light responsive elements, hormone-responsive elements and stress-responsive elements, etc. Seven fragments including the full-length promoter and six 5′ terminal series deleted fragments were fused with the GUS reporter gene to test their activities. The results of histochemical staining show that a strong GUS activity were observed in flowers, siliques, leaves, stems and roots of transgenic Arabidopsis containing the full length Pq3-O-UGT2 promoter. Different GUS activity were also observed in the seedlings of transgenic Arabidopsis containing the full length promoter and six 5′ terminal series deleted fragments. Fluorometric assays show that seven fragments were found to drive GUS expression, and the highest enzyme activity is the full-length fragment with 4370 pmol 4-MU/min/mg protein, which is 80.01% of the CaMV35S promoter. Followed by P-801::GUS with 2162 pmol 4-MU/min/mg protein, and the shortest promoter containing P-198::GUS with 45 pmol 4-MU/min/mg protein was sufficient to activate GUS expression. In addition, extended light, low temperatures, Methyl jasmonate(MeJA), Abscisic acid(ABA), NAA and GA3 were selected to investigate the Pq3-O-UGT2 promoter in response to abiotic stress and hormone treatment. The promoter activity of the full length can be enhanced much more than the other six 5′ terminal series deleted fragments, and the most significant change was detected in MeJA treatment with 2.12 times increased. Furthermore, it was found that the promoter activity of P-998::GUS can be enhanced by ABA with 1.47 times. Above results show that the GUS activity of different promoter fragments had different response to different environmental factors. This article provides a great understanding of complex regulatory mechanisms of Pq3-O-UGT2 and the molecular mechanisms of triterpene biosynthesis.