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

Abstract

Pq3-O-UGT2 is a key UDP-glycosyltransferase gene in the ginsenoside biosynthesis pathway of Panax quinquefolium L. Functional analysis of promoter sequences plays an important role in understanding the regulation of functional gene expression. In this study, chromosome walking technology was used to isolate the 1399 bp sequence upstream of the ATG initiation codon of Pq3-O-UGT2. Bioinformatics analysis shows that the promoter sequence contains a large number of putative cis-acting elements responsive to exogenous and endogenous factors. The full-length promoter and six 5′ terminal truncations were fused with the GUS reporter gene to test their activities. The results of histochemical staining showed 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. Fluorometric assays showed that the highest enzyme activity is the full-length promoter with 4370 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, MeJA, ABA, NAA and GA3 were selected to investigate the Pq3-O-UGT2 promoter in response to abiotic stress and hormone treatment. The GUS activity of Pq3-O-UGT2 full-length promoter (P-1399) was 2.12 times that of the control plant after MeJA treatment, suggesting that P-1399 is a MeJA-inducible promoter. Furthermore, the assay results showed that ABA could extensively induce GUS expression in five 5′ truncated promoter transgenic plants, except P-198. These experimental results will help us to better explore the regulatory mechanisms in the promoter region of the Pq3-O-UGT2 gene and contribute to further studies of the molecular mechanisms of glycosylation in ginseng plants. The Pq3-O-UGT2 promoter was successfully cloned. Seven 5′ truncations could drive the expression of GUS gene in transgenic Arabidopsis plants and the full-length promoter is a MeJA-inducible promoter.