Functional analysis of the promoter of a UDP-glycosyltransferase gene from Panax quinquefolius
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Glycosyltransferases, a multigene superfamily in plants, are necessary to synthesize, modify and diversify specific ginsenosides in Panax quinquefolius. P. quinquefolius is widely used in medicine and nutrition. Various glycosyltransferases catalyze glycosylation to modify the pharmacological and biological activity of ginsenosides in ginseng plants. Two UDP-glycosyltransferase genes in P. quinquefolius, Pq3-O-UGT1 and Pq3-O-UGT2 (Genbank accession Nos. KR028477, KR106207), have been isolated and identified, but the signal transduction and transcriptional control mechanisms of glycosyltransferase genes have not yet been fully identified. To understand the expression and regulatory mechanism of Pq3-O-UGT1, we isolated a 2,611-bp upstream sequence of Pq3-O-UGT1 gene from P. quinquefolius using genome walking method. The result of sequence analysis indicated Pq3-O-UGT1 promoter included many essential putative cis-elements that may be responsible for the spatial and temporal expression. The full-length promoter fragment and its 5′-deletions were merged with the β-glucuronidase (GUS) reporter gene and transferred into tobacco plants to test their activities. The results of histochemical staining and fluorometric determination indicated that the full-length promoter was found to induce GUS expression preferentially in tender leaf, bud, petiole and stem with much lower activity than the cauliflower mosaic virus 35S promoter. Moreover, promoter deletion analysis revealed that the minimal promoter containing 487-bp fragment was sufficient to strongly activate GUS expression. The promoter activity of P-487 (7.6 nmol MU/min/µg protein) was approximately 90 times more than that of full-length fragment. Furthermore, it was found that the promoter activity can be enhanced by SA, GA, NAA and the expression of P-2246 was enhanced by light, but insignificant change was detected in drought treatment. These findings will help us to better understand the regulatory mechanisms of the upstream region of the Pq3-O-UGT1 gene and provide useful information for further investigation of the molecular mechanisms of glycosylation in ginseng plants.
KeywordsFunction analysis Promoter Transgenic tobacco Glycosyltransferase Panax quinquefolius
This work was supported by National High Technology Research and Development Program of China (863), No. 2013AA102604. Projects of National Science Foundation of China, No. 31270337. Research Fund for the Doctoral Program of Higher Education of China, No. 20120061110038 and Scientific and Technological Development Plan Project of Jilin Province, No. 20130102041JC.
This research was accomplished with the collaboration of all authors. CL was responsible for the major work including isolation of Pq3-O-UGT1 promoter, functional analysis, the data processing work and wrote part of the manuscript. SZ was responsible for the experimental guidance and provision of equipment. PF and XW were responsible for the data analysis and wrote part of the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
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