Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 135, Issue 3, pp 381–393 | Cite as

Functional analysis of the promoter of a UDP-glycosyltransferase gene from Panax quinquefolius

  • Chao Lu
  • Shou-jing ZhaoEmail author
  • Peng-cheng Feng
  • Xue-song Wang
Original Article


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.


Function 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.

Author contributions

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.


  1. Ahrazem O, Rubio-Moraga A, Mozos AT, Gómez-Gómez ML (2014) Genomic organization of a UDP-glucosyltransferase gene determines differential accumulation of specific flavonoid glucosides in tepals. Plant Cell Tissue Organ Cult 119(2):227–245CrossRefGoogle Scholar
  2. Ali MB, Yu K-W, Hahn E-J, Paek K-Y (2006) Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors. Plant Cell Rep 25(6):613–620. CrossRefPubMedGoogle Scholar
  3. Anders N, Wilkinson MD, Lovegrove A, Freeman J, Tryfona T, Pellny TK, Weimar T, Mortimer JC, Stott K, Baker JM, Defoin-Platel M, Shewry PR, Dupree P, Mitchell RAC (2012) Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses. Proc Natl Acad Sci USA 109(3):989–993. CrossRefPubMedGoogle Scholar
  4. Augustin JM, Kuzina V, Andersen SB, Bak S (2011) Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry 72(6):435–457. CrossRefPubMedGoogle Scholar
  5. Bhuria M, Goel P, Kumar S, Singh AK (2016) The promoter of AtUSP is co-regulated by phytohormones and abiotic stresses in Arabidopsis thaliana. Front Plant Sci. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Biswas T, Kalra A, Mathur AK, Lal RK, Singh M, Mathur A (2016) Elicitors’ influenced differential ginsenoside production and exudation into medium with concurrent Rg3/Rh2 panaxadiol induction in Panax quinquefolius cell suspensions. Appl Microbiol Biotechnol 100(11):4909–4922. CrossRefPubMedGoogle Scholar
  7. Chen W, Kui L, Zhang G, Zhu S, Zhang J, Wang X, Yang M, Huang H, Liu Y, Wang Y, Li Y, Zeng L, Wang W, He X, Dong Y, Yang S (2017) Whole-genome sequencing and analysis of the Chinese herbal plant Panax notoginseng. Mol Plant 10(6):899–902. CrossRefPubMedGoogle Scholar
  8. Dalal M, Chinnusamy V, Bansal KC (2010) Isolation and functional characterization of lycopene beta-cyclase (CYC-B) promoter from Solanum habrochaites. BMC Plant Biol 10(1):61–61. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Dong W, Niu L, Li H, Gao F (2016) Isolation and analysis of the promoter of IbMYB1 gene from storage roots of purple-fleshed sweet potato. J Plant Biochem Biotechnol 25(3):278–284. CrossRefGoogle Scholar
  10. Ha LT, Pawlicki-Jullian N, Pillon-Lequart M, Boitel-Conti M, Duong HX, Gontier E (2016) Hairy root cultures of Panax vietnamensis, a promising approach for the production of ocotillol-type ginsenosides. Plant Cell Tissue Organ Cult 126(1):93–103. CrossRefGoogle Scholar
  11. Han JY, Kwon YS, Yang DC, Jung YR, Choi YE (2006) Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng. Plant Cell Physiol 47(12):1653–1662. CrossRefPubMedGoogle Scholar
  12. Hu M, Lu Z, Guo J, Luo Y, Li H, Li L, Gao F (2016) Cloning and characterization of the cDNA and promoter of UDP-glucose: flavonoid 3-O-glucosyltransferase gene from a purple-fleshed sweet potato. S Afr J Bot 106:211–220. CrossRefGoogle Scholar
  13. Huang C, Qian ZG, Zhong JJ (2013) Enhancement of ginsenoside biosynthesis in cell cultures of Panax ginseng by N,N ‘-dicyclohexylcarbodiimide elicitation. J Biotechnol 165(1):30–36. CrossRefPubMedGoogle Scholar
  14. Hue HTT, Ha DTT, Hai NV, Hien LTT (2016) Isolation and characterization of the 4-coumarate:coenzyme A ligase (4CL1) promoter from Eucalyptus camaldulensis. Physiol Mol Biol Plants 22(3):399–405. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jeong C-S, Murthy HN, Hahn E-J, Paek K-Y (2008) Improved production of ginsenosides in suspension cultures of ginseng by medium replenishment strategy. J Biosci Bioeng 105(3):288–291. CrossRefPubMedGoogle Scholar
  16. Jiang YJ, Piao XC, Liu JS, Jiang J, Lian ZX, Kim MJ, Lian ML (2015) Bioactive compound production by adventitious root culture of Oplopanax elatus in balloon-type airlift bioreactor systems and bioactivity property. Plant Cell Tissue Organ Cult 123(2):413–425. CrossRefGoogle Scholar
  17. Jiang M, Liu J, Quan X, Quan L, Wu S (2016) Different chilling stresses stimulated the accumulation of different types of ginsenosides in Panax ginseng cells. Acta Physiol Plant 38(8):1–8. CrossRefGoogle Scholar
  18. Jiu S, Wang C, Zheng T, Liu Z, Leng X, Pervaiz T, Lotfi A, Fang J, Wang X (2016) Characterization of VvPAL-like promoter from grapevine using transgenic tobacco plants. Funct Integr Genomics 16(6):595–617. CrossRefPubMedGoogle Scholar
  19. Jung S-C, Kim W, Park SC, Jeong J, Park MK, Lim S, Lee Y, Im W-T, Lee JH, Choi G, Kim SC (2014) Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg(3) and Rd. Plant Cell Physiol 55(12):2177–2188. CrossRefPubMedGoogle Scholar
  20. Khorolragchaa A, Kim YJ, Rahimi S, Sukweenadhi J, Jang MG, Yang DC (2014) Grouping and characterization of putative glycosyltransferase genes from Panax ginseng Meyer. Gene 536(1):186–192. CrossRefPubMedGoogle Scholar
  21. Kim OT, Bang KH, Kim YC, Hyun DY, Kim MY, Cha SW (2009) Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate. Plant Cell Tissue Organ Cult 98(1):25–33. CrossRefGoogle Scholar
  22. Kim T-D, Han J-Y, Huh GH, Choi Y-E (2011) Expression and functional characterization of three squalene synthase genes associated with saponin biosynthesis in Panax ginseng. Plant Cell Physiol 52(1):125–137. CrossRefPubMedGoogle Scholar
  23. Kim OT, Yoo NH, Kim GS, Kim YC, Bang KH, Hyun DY, Kim SH, Kim MY (2013) Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate. Plant Cell Tissue Organ Cult 112(1):87–93. CrossRefGoogle Scholar
  24. Kim Y-J, Lee OR, Oh JY, Jang M-G, Yang D-C (2014) Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding genes in triterpene saponin-producing ginseng. Plant Physiol 165(1):373–387. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kim YJ, Zhang DB, Yang DC (2015) Biosynthesis and biotechnological production of ginsenosides. Biotechnol Adv 33(6):717–735. CrossRefPubMedGoogle Scholar
  26. Lee J, Mudge KW (2013) Water deficit affects plant and soil water status, plant growth, and ginsenoside contents in American ginseng. Hortic Environ Biotechnol 54(6):475–483. CrossRefGoogle Scholar
  27. Li Y, Sun Y, Yang Q, Kang J, Zhang T, Gruber MY, Fang F (2012) Cloning and function analysis of an alfalfa (Medicago sativa L.) zinc finger protein promoter MsZPP. Mol Biol Rep 39(8):8559–8569. CrossRefPubMedGoogle Scholar
  28. Li Y, Liu X, Li J, Li S, Chen G, Zhou X, Yang W, Chen R (2015) Isolation of a maize ZmCI-1B promoter and characterization of its activity in transgenic maize and tobacco. Plant Cell Rep 34(8):1443–1457. CrossRefPubMedGoogle Scholar
  29. Liang Y, Zhao S (2008) Progress in understanding of ginsenoside biosynthesis. Plant Biol 10(4):415–421. CrossRefPubMedGoogle Scholar
  30. Liang Y, Wu J, Li Y, Li J, Ouyang Y, He Z, Zhao S (2015) Enhancement of ginsenoside biosynthesis and secretion by Tween 80 in Panax ginseng hairy roots. Biotechnol Appl Biochem 62(2):193–199. CrossRefPubMedGoogle Scholar
  31. Lim W, Mudge KW, Lee JW (2006) Effect of water stress on ginsenoside production and growth of American ginseng. HortTechnology 16(3):517–522Google Scholar
  32. Lim S-H, Kim JK, Lee J-Y, Kim Y-M, Sohn S-H, Kim D-H, Ha S-H (2013) Petal-specific activity of the promoter of an anthocyanidin synthase gene of tobacco (Nicotiana tabacum L.). Plant Cell Tissue Organ Cult (PCTOC) 114(3):373–383. CrossRefGoogle Scholar
  33. Lu S, Zhang Y, Zheng X, Zhu K, Xu Q, Deng X (2016) Isolation and functional characterization of a lycopene β-cyclase gene promoter from citrus. Front Plant Sci. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lu C, Zhao S-J, Wang X-S (2017a) Functional regulation of a UDP-glucosyltransferase gene (Pq3-O-UGT1) by RNA interference and overexpression in Panax quinquefolius. Plant Cell Tissue Organ Cult 129(3):445–456. CrossRefGoogle Scholar
  35. Lu C, Zhao S, Wei G, Zhao H, Qu Q (2017b) Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius. Plant Physiol Biochem 111:67–76CrossRefGoogle Scholar
  36. Lv D, Zhang Y (2016) Isolation and functional analysis of apple MdHMGR1 and MdHMGR4 gene promoters in transgenic Arabidopsis thaliana. Plant Cell Tissue Organ Cult 129(1):133–143. CrossRefGoogle Scholar
  37. Pace R, Martinelli EM, Sardone N, Combarieu EDE (2015) Metabolomic evaluation of ginsenosides distribution in Panax genus (Panax ginseng and Panax quinquefolius) using multivariate statistical analysis. Fitoterapia 101:80–91. CrossRefPubMedGoogle Scholar
  38. Parvathy ST, Srinivasan R (2016) Functional analysis of a cryptic promoter from Arabidopsis thaliana reveals bidirectionality. Plant Biotechnol Rep 10(4):241–255. CrossRefGoogle Scholar
  39. Qi L-W, Wang C-Z, Yuan C-S (2011) Ginsenosides from American ginseng: chemical and pharmacological diversity. Phytochemistry 72(8):689–699. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Rahimi S, Kim YJ, Yang DC (2015) Production of ginseng saponins: elicitation strategy and signal transductions. Appl Microbiol Biotechnol 99(17):6987–6996. CrossRefPubMedGoogle Scholar
  41. Rai A, Yamazaki M, Takahashi H, Nakamura M, Kojoma M, Suzuki H, Saito K (2016) RNA-seq transcriptome analysis of Panax japonicus, and Its comparison with other Panax species to identify potential genes involved in the saponins biosynthesis. Front Plant Sci 7:e0144Google Scholar
  42. Schlag EM, McIntosh MS (2013) The relationship between genetic and chemotypic diversity in American ginseng (Panax quinquefolius L.). Phytochemistry 93:96–104. CrossRefPubMedGoogle Scholar
  43. Seki H, Tamura K, Muranaka T (2015) P450s and UGTs: key players in the structural diversity of triterpenoid saponins. Plant Cell Physiol 56(8):1463–1471. CrossRefPubMedGoogle Scholar
  44. Smirnova YN, Reshetnyak OV, Smolenskaya IN, Voevudskaya SY, Nosov AM (2010) Effect of growth regulators on ginsenoside production in the cell culture of two ginseng species. Russ J Plant Physiol 57(3):430–437. CrossRefGoogle Scholar
  45. Sun Y, Zhao S-J, Liang Y-L, Le W, Cao H-J (2013) Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences. Plant Growth Regul 71(3):207–217. CrossRefGoogle Scholar
  46. Tang X, Gan XT, Rajapurohitam V, Huang CX, Xue J, Lui EMK, Karmazyn M (2016) North American ginseng (Panax quinquefolius) suppresses beta-adrenergic-dependent signalling, hypertrophy, and cardiac dysfunction. Can J Physiol Pharmacol 94(12):1325–1335. CrossRefPubMedGoogle Scholar
  47. Wang W, Zhang Z-Y, Zhong J-J (2005) Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation. Appl Microbiol Biotechnol 67(6):752–758. CrossRefPubMedGoogle Scholar
  48. Wang L, Zhao S-J, Cao H-J, Sun Y (2014a) The isolation and characterization of dammarenediol synthase gene from Panax quinquefolius and its heterologous co-expression with cytochrome P450 gene PqD12H in yeast. Funct Integr Genomics 14(3):545–557. CrossRefPubMedGoogle Scholar
  49. Wang L, Zhao S-J, Liang Y-L, Sun Y, Cao H-J, Han Y (2014b) Identification of the protopanaxatriol synthase gene CYP6H for ginsenoside biosynthesis in Panax quinquefolius. Funct Integr Genomics 14(3):559–570. CrossRefPubMedGoogle Scholar
  50. Wang P, Wei Y, Fan Y, Liu Q, Wei W, Yang C, Zhang L, Zhao G, Yue J, Yan X, Zhou Z (2015) Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts. Metab Eng 29:97–105. CrossRefPubMedGoogle Scholar
  51. Washida D, Shimomura K, Takido M, Kitanaka S (2004) Auxins affected ginsenoside production and growth of hairy roots in Panax hybrid. Biol Pharm Bull 27(5):657–660. CrossRefPubMedGoogle Scholar
  52. Wei W, Wang P, Wei Y, Liu Q, Yang C, Zhao G, Yue J, Yan X, Zhou Z (2015) Characterization of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts. Mol Plant 8(9):1412–1424. CrossRefPubMedGoogle Scholar
  53. Xin S, Tao CC, Li HB (2016) Cloning and functional analysis of the promoter of an ascorbate oxidase gene from Gossypium hirsutum. PLoS ONE 11(9):e0161695. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Xiu H, Nuruzzaman M, Guo XQ, Cao HZ, Huang JJ, Chen XH, Wu KL, Zhang R, Huang YZ, Luo JL, Luo ZY (2016) Molecular cloning and expression analysis of eight PgWRKY genes in Panax ginseng responsive to salt and hormones. Int J Mol Sci 17(3):319. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Yan X, Fan Y, Wei W, Wang P, Liu Q, Wei Y, Zhang L, Zhao G, Yue J, Zhou Z (2014) Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 24(6):770–773. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Yang Q, Yuan D, Shi L, Capell T, Bai C, Wen N, Lu X, Sandmann G, Christou P, Zhu C (2012) Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants. Transgenic Res 21(5):1043–1056. CrossRefPubMedGoogle Scholar
  57. Yonekura-Sakakibara K, Hanada K (2011) An evolutionary view of functional diversity in family 1 glycosyltransferases. Plant J 66(1):182–193. CrossRefPubMedGoogle Scholar
  58. Yu K-W, Murthy HN, Hahn E-J, Paek K-Y (2005) Ginsenoside production by hairy root cultures of Panax ginseng: influence of temperature and light quality. Biochem Eng J 23(1):53–56. CrossRefGoogle Scholar
  59. Zhang D, Li W, Xia E-h, Zhang Q-j, Liu Y, Zhang Y, Tong Y, Zhao Y, Niu Y-c, Xu J-h, Gao L-z (2017) The medicinal herb Panax notoginseng genome provides insights into ginsenoside biosynthesis and genome evolution. Mol Plant 10(6):903–907. CrossRefPubMedGoogle Scholar
  60. Zhao CL, Cui XM, Chen YP, Liang Q (2010) Key enzymes of triterpenoid saponin biosynthesis and the induction of their activities and gene expressions in plants. Nat Prod Commun 5(7):1147–1158PubMedGoogle Scholar
  61. Zhu C, Yang Q, Ni X, Bai C, Sheng Y, Shi L, Capell T, Sandmann G, Christou P (2014) Cloning and functional analysis of the promoters that upregulate carotenogenic gene expression during flower development in Gentiana lutea. Physiol Plant 150(4):493–504. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Chao Lu
    • 1
    • 2
  • Shou-jing Zhao
    • 1
    • 3
    Email author
  • Peng-cheng Feng
    • 3
  • Xue-song Wang
    • 3
  1. 1.School of Biological and Agricultural EngineeringJilin UniversityChangchunPeople’s Republic of China
  2. 2.School of Life SciencesZhengzhou Normal UniversityZhengzhouPeople’s Republic of China
  3. 3.School of Life SciencesJilin UniversityChangchunPeople’s Republic of China

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