Selection and validation of reference genes desirable for gene expression analysis by qRT-PCR in MeJA-treated ginseng hairy roots

Autoři: Li Li aff001;  Kangyu Wang aff001;  Mingzhu Zhao aff001;  Shaokun Li aff001;  Yue Jiang aff001;  Lei Zhu aff001;  Jing Chen aff001;  Yanfang Wang aff002;  Chunyu Sun aff001;  Ping Chen aff001;  Jun Lei aff001;  Meiping Zhang aff001;  Yi Wang aff001
Působiště autorů: College of Life Science, Jilin Agricultural University, Changchun, Jilin, China aff001;  Research Center of Ginseng Genetic Resources Development and Utilization, Changchun, Jilin, China aff002;  College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun, Jilin, China aff003;  Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules (Yanbian University), Ministry of Education, Yanji, Jilin, China aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


Ginseng is a valuable herb of traditional Chinese medicine and ginsenosides, the main bioactive components of ginseng, have been proven to have multiple functions in human therapies and health. Methyl jasmonate (MeJA) is an elicitor that has been demonstrated to have a vital influence on ginsenoside biosynthesis. Quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in quantification of gene expressions. Here, we report the selection and validation of reference genes desirable for normalization of gene expressions quantified by qRT-PCR in ginseng hairy roots treated with MeJA. Twelve reference genes were selected as candidate genes, and their expressions were quantified by qRT-PCR, and analyzed by geNorm, NormFinder and BestKeeper. CYP and EF-1α were shown to be the most stable reference genes in geNorm, CYP was the most stable reference gene in NormFinder, and 18S was the most stable reference gene in BestKeeper. On this basis, we further quantified the relative expression levels of four genes encoding key enzymes that are involved in ginsenoside biosynthesis using CYP and 18S as the reference genes, respectively. Moreover, correlation analysis was performed between the quantified expressions of four genes and the ginsenoside content in MeJA-treated ginseng hairy roots. The results of relative expressions of the four genes quantified using CYP as the reference gene and their significant correlations with the ginsenoside content were better than those using 18S as the reference gene. The CYP gene, hence, was concluded as the most desirable reference gene for quantification of the expressions of genes in MeJA-treated ginseng hairy roots. This finding, therefore, provides information useful for gene research in ginseng, particularly in MeJA-treated ginseng hairy roots, which includes identification and characterization of genes involved in ginsenoside biosynthesis.

Klíčová slova:

Biosynthesis – Gene expression – Hair – Polymerase chain reaction – RNA extraction – Seedlings – Glycosides – Secondary metabolism


1. Ahuja A, Kim JH, Kim J-H, Yi Y-S, Cho JY. Functional role of ginseng-derived compounds in cancer. Journal of ginseng research. 2018;42(3):248–254. doi: 10.1016/j.jgr.2017.04.009 29983605

2. Park SB, Park GH, Um Y, Kim HN, Song HM, Kim N, et al. Wood-cultivated ginseng exerts anti-inflammatory effect in LPS-stimulated RAW264. 7 cells. International journal of biological macromolecules. 2018;116:327–334. doi: 10.1016/j.ijbiomac.2018.05.039 29751039

3. Kim J-H. Pharmacological and medical applications of Panax ginseng and ginsenosides: A review for use in cardiovascular diseases. Journal of ginseng research. 2018;42(3):264–269. doi: 10.1016/j.jgr.2017.10.004 29983607

4. Kim KH, Lee D, Lee HL, Kim C-E, Jung K, Kang KS. Beneficial effects of Panax ginseng for the treatment and prevention of neurodegenerative diseases: past findings and future directions. Journal of ginseng research. 2018;42(3):239–247. doi: 10.1016/j.jgr.2017.03.011 29989012

5. Patel S, Rauf A. Adaptogenic herb ginseng (Panax) as medical food: Status quo and future prospects. Biomedicine & Pharmacotherapy. 2017;85:120–127.

6. Adil M, Jeong BR. In vitro cultivation of Panax ginseng C.A. Meyer. Industrial crops and products. 2018;122:239–251.

7. Zhang T, Zhong S, Hou L, Wang Y, Xing X, Guan T, et al. Computational and experimental characterization of estrogenic activities of 20 (S, R)-protopanaxadiol and 20 (S, R)-protopanaxatriol. Journal of Ginseng Research. 2018.

8. Kim Y-J, Zhang D, Yang D-C. Biosynthesis and biotechnological production of ginsenosides. Biotechnology advances. 2015;33(6):717–735.

9. Zhang Y-C, Li G, Jiang C, Yang B, Yang H-J, Xu H-Y, et al. Tissue-specific distribution of ginsenosides in different aged ginseng and antioxidant activity of ginseng leaf. Molecules. 2014;19(11):17381–17399. doi: 10.3390/molecules191117381 25353387

10. Li J, Liu S, Wang J, Li J, Liu D, Li J, et al. Fungal elicitors enhance ginsenosides biosynthesis, expression of functional genes as well as signal molecules accumulation in adventitious roots of Panax ginseng C.A. Mey. Journal of biotechnology. 2016;239:106–114. doi: 10.1016/j.jbiotec.2016.10.011 27746309

11. Huang C, Qian Z-G, Zhong J-J. Enhancement of ginsenoside biosynthesis in cell cultures of Panax ginseng by N, N′-dicyclohexylcarbodiimide elicitation. Journal of biotechnology. 2013;165(1):30–36. doi: 10.1016/j.jbiotec.2013.02.012 23467002

12. Tewari RK, Paek K-Y. Salicylic acid-induced nitric oxide and ROS generation stimulate ginsenoside accumulation in Panax ginseng roots. Journal of plant growth regulation. 2011;30(4):396–404.

13. Vasconsuelo A, Boland R. Molecular aspects of the early stages of elicitation of secondary metabolites in plants. Plant Science. 2007;172(5):861–875.

14. Yu K-W, Gao W-Y, Son S-H, Paek K-Y. Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C.A. Meyer). In Vitro Cellular & Developmental Biology-Plant. 2000;36(5):424–428.

15. Palazón J, Cusidó RM, Bonfill M, Mallol A, Moyano E, Morales C, et al. Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production. Plant Physiology and Biochemistry. 2003;41(11–12):1019–1025.

16. Ali MB, Yu K-W, Hahn E-J, Paek K-Y. Differential responses of anti-oxidants enzymes, lipoxygenase activity, ascorbate content and the production of saponins in tissue cultured root of mountain Panax ginseng CA Mayer and Panax quinquefolium L. in bioreactor subjected to methyl jasmonate stress. Plant science. 2005;169(1):83–92.

17. Cao H, Nuruzzaman M, Xiu H, Huang J, Wu K, Chen X, et al. Transcriptome analysis of methyl jasmonate-elicited Panax ginseng adventitious roots to discover putative ginsenoside biosynthesis and transport genes. International journal of molecular sciences. 2015;16(2):3035–3057. doi: 10.3390/ijms16023035 25642758

18. Oh JY, Kim Y-J, Jang M-G, Joo SC, Kwon W-S, Kim S-Y, et al. Investigation of ginsenosides in different tissues after elicitor treatment in Panax ginseng. Journal of ginseng research. 2014;38(4):270–277. doi: 10.1016/j.jgr.2014.04.004 25379007

19. Udvardi MK, Czechowski T, Scheible W-R. Eleven golden rules of quantitative RT-PCR. The Plant Cell. 2008;20(7):1736–1737. doi: 10.1105/tpc.108.061143 18664613

20. Chapman JR, Waldenström J. With reference to reference genes: a systematic review of endogenous controls in gene expression studies. PloS one. 2015;10(11):e0141853. doi: 10.1371/journal.pone.0141853 26555275

21. Liu J, Wang Q, Sun M, Zhu L, Yang M, Zhao Y. Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs. PloS one. 2014;9(11):e112177. doi: 10.1371/journal.pone.0112177 25393243

22. Wang M, Lu S. Validation of suitable reference genes for quantitative gene expression analysis in Panax ginseng. Frontiers in plant science. 2016;6:1259. doi: 10.3389/fpls.2015.01259 26793228

23. Chi C, Shen Y, Yin L, Ke X, Han D, Zuo Y. Selection and validation of reference genes for gene expression analysis in Vigna angularis using quantitative real-time RT-PCR. PloS one. 2016;11(12):e0168479. doi: 10.1371/journal.pone.0168479 27992593

24. Hu Y, Deng T, Chen L, Wu H, Zhang S. Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata. PloS one. 2016;11(4):e0154384. doi: 10.1371/journal.pone.0154384 27124298

25. Reddy DS, Bhatnagar-Mathur P, Reddy PS, Cindhuri KS, Ganesh AS, Sharma KK. Identification and validation of reference genes and their impact on normalized gene expression studies across cultivated and wild cicer species. PloS one. 2016;11(2):e0148451. doi: 10.1371/journal.pone.0148451 26863232

26. Chang Y-W, Chen J-Y, Lu M-X, Gao Y, Tian Z-H, Gong W-R, et al. Selection and validation of reference genes for quantitative real-time PCR analysis under different experimental conditions in the leafminer Liriomyza trifolii (Diptera: Agromyzidae). PloS one. 2017;12(7):e0181862. doi: 10.1371/journal.pone.0181862 28746411

27. Dai T-M, Lü Z-C, Liu W-X, Wan F-H. Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED. PloS one. 2017;12(3):e0173821. doi: 10.1371/journal.pone.0173821 28323834

28. Liu Y, Liu J, Xu L, Lai H, Chen Y, Yang Z, et al. Identification and validation of reference genes for seashore paspalum response to abiotic stresses. International journal of molecular sciences. 2017;18(6):1322.

29. Yue Y, Qiu Z-D, Qu X-Y, Deng A-P, Yuan Y, Huang L-Q. Discoursing on Soxhlet extraction of ginseng using association analysis and scanning electron microscopy. Journal of pharmaceutical analysis. 2018;8(5):312–317. doi: 10.1016/j.jpha.2018.08.003 30345145

30. Yue-Yi C JS, En Y U, et al. Study on transformation of ginsenoside Rg_3 fermented by Monascus purpureus. Chinese Traditional and Herbal Drugs. 2018. (In Chinese)

31. Yu-Nan Z, Zhong-Li W, Jian-Guo D, Lin C, HUANG Y-F. Preparation and quality assessment of high-purity ginseng total saponins by ion exchange resin combined with macroporous adsorption resin separation. Chinese journal of natural medicines. 2014;12(5):382–392. doi: 10.1016/S1875-5364(14)60048-0 24856763

32. Kim O, Bang K, Jung S, Kim Y, Hyun D, Kim S, et al. Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate. Biologia plantarum. 2010;54(1):47–53.

33. Han JY, Kwon YS, Yang DC, Jung YR, Choi YE. Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng. Plant and cell physiology. 2006;47(12):1653–1662. doi: 10.1093/pcp/pcl032 17088293

34. Jung S-C, Kim W, Park SC, Jeong J, Park MK, Lim S, et al. Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd. Plant and Cell Physiology. 2014;55(12):2177–2188. doi: 10.1093/pcp/pcu147 25320211

35. Han J-Y, Kim H-J, Kwon Y-S, Choi Y-E. The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant and cell physiology. 2011;52(12):2062–2073. doi: 10.1093/pcp/pcr150 22039120

36. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome biology. 2002;3(7):research0034. 1.

37. Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer research. 2004;64(15):5245–5250. doi: 10.1158/0008-5472.CAN-04-0496 15289330

38. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnology letters. 2004;26(6):509–515. doi: 10.1023/b:bile.0000019559.84305.47 15127793

39. Kim Y-S, Hahn E-J, Murthy HN, Paek K-y. Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate. Biotechnology Letters. 2004;26(21):1619–1622. doi: 10.1007/s10529-004-3183-2 15604808

40. Kang KB, Jayakodi M, Lee YS, Nguyen VB, Park H-S, Koo HJ, et al. Identification of candidate UDP-glycosyltransferases involved in protopanaxadiol-type ginsenoside biosynthesis in Panax ginseng. Scientific reports. 2018;8(1):11744. doi: 10.1038/s41598-018-30262-7 30082711

41. Verma N, Shukla S. Impact of various factors responsible for fluctuation in plant secondary metabolites. Journal of Applied Research on Medicinal and Aromatic Plants. 2015;2(4):105–113.

42. Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, et al. JAZ repressor proteins are targets of the SCF COI1 complex during jasmonate signalling. Nature. 2007;448(7154):661. doi: 10.1038/nature05960 17637677

43. Wasternack C, Strnad M. Jasmonates are signals in the biosynthesis of secondary metabolites—Pathways, transcription factors and applied aspects—A brief review. New biotechnology. 2019;48:1–11. doi: 10.1016/j.nbt.2017.09.007 29017819

44. Per TS, Khan MIR, Anjum NA, Masood A, Hussain SJ, Khan NA. Jasmonates in plants under abiotic stresses: Crosstalk with other phytohormones matters. Environmental and experimental botany. 2018;145:104–120.

45. Shen Q, Lu X, Yan T, Fu X, Lv Z, Zhang F, et al. The jasmonate‐responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytologist. 2016;210(4):1269–1281. doi: 10.1111/nph.13874 26864531

46. Lu C, Zhao S, Wei G, Zhao H, Qu Q. Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius. Plant physiology and biochemistry. 2017;111:67–76. doi: 10.1016/j.plaphy.2016.11.017 27914321

47. Subramaniyam S, Mathiyalagan R, Natarajan S, Kim Y-J, Jang M-g, Park J-H, et al. Transcript expression profiling for adventitious roots of Panax ginseng Meyer. Gene. 2014;546(1):89–96. doi: 10.1016/j.gene.2014.05.024 24831831

48. Zhang R, Zhu J, Cao H-Z, An Y-R, Huang J-J, Chen X-H, et al. Molecular cloning and expression analysis of PDR1-like gene in ginseng subjected to salt and cold stresses or hormonal treatment. Plant physiology and biochemistry. 2013;71:203–211. doi: 10.1016/j.plaphy.2013.07.011 23968928

49. Park S-B, Chun J-H, Ban Y-W, Han JY, Choi YE. Alteration of Panax ginseng saponin composition by overexpression and RNA interference of the protopanaxadiol 6-hydroxylase gene (CYP716A53v2). Journal of ginseng research. 2016;40(1):47–54. doi: 10.1016/j.jgr.2015.04.010 26843821

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden