Abstract
Bupleurum chinense DC. is one of the source plants of a well-known crude drug, Chai hu (Radix Bupleuri), producing triterpenoid saponins (saikosaponins) with a wide-spectrum of pharmacological applications. The biosynthesis of triterpenoid saponins involved with the cyclizing of the precursor 2,3-oxidosqualene to produce the first committed triterpene β-amyrin catalyzed by β-amyrin synthase (β-AS), whereafter diverse of triterpenoid saponins was biosynthesized. In addition, 2,3-oxidosqualene could be catalyzed by cycloartenol synthase directing to the synthesis of phytosterol. β-AS was thus defined as an important branch point between primary and secondary metabolisms, and may play a regulating role in the control of triterpenoid saponins biosynthesis. In this study, the promoter and protein-encoding regions of a β-AS gene (designated bcAS1) were isolated by genome walking and PCR from B. chinense. Several important cis-acting elements for gene regulation were identified within the promoter region including light-responsive, hormone-responsive and various other stress-related elements. Approximate 0.8 kb fragment on upstream of ATG start codon of bcAS1 was sub-cloned into pAN580 vector to replace the 35S promoter driving the expression of green fluorescent protein (GFP) gene. The promoter activity was detected by transient expression in onion epidermis cells by the expression of GFP. Approximately 6 kb length of bcAS1 gene was cloned, containing 18 exons and 17 introns. Although a dozen of β-AS cDNA was isolated, seldom the promoter and gene of it was reported. This work was a valuable foundation for further studies on the regulatory role of β-AS in biosynthesis of saikosaponins.
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References
Ashour ML, Wink M (2011) Genus Bupleurum: a review of its phytochemistry, pharmacology and modes of action. J Pharm Pharmacol 63:305–321
Cammareri M, Consiglio MF, Pecchia P, Corea G, Lanzotti V, Ibeas JI, Tava A, Conicella C (2008) Molecular characterization of β-amyrin synthase from Aster sedifolius L. and triterpenoid saponin analysis. Plant Sci 175:255–261
Confalonieri M, Cammareri M, Biazzi E, Pecchia P, Fevereiro MPS, Balestrazzi A, Tava A, Conicella C (2009) Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel β-amyrin synthase (AsOXA1) gene. Plant Biotechnol J 7:172–182
Dai ZB, Liu Y, Guo J, Huang LQ, Zhang XL (2015) Yeast synthetic biology for high-value metabolites. FEMS Yeast Res 15:1–11
Dhar N, Rana S, Razdan S, Bhat WW, Hussain A, Dhar RS, Vaishnavi S, Hamid A, Vishwakarma R, Lattoo SK (2014) Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal. J Biol Chem 289:17249–17267
Hayashi H, Huang P, Kirakosyan A, Inoue K, Hiraoka N, Ikeshiro Y, Kushiro T, Shibuya M, Ebizuka Y (2001) Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice. Biol Pharm Bull 24:912–916
Hayashi H, Huang PY, Inoue K (2003) Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell Physiol 44:404–411
Hayashi H, Huang PY, Takada S, Obinata M, Inoue K, Shibuya M, Ebizuka Y (2004) Differential expression of three oxidosqualene cyclase mRNAs in Glycyrrhiza glabra. Biol Pharm Bull 27:1086–1092
Iturbe-Ormaetxe I, Haralampidis K, Papadopoulou K, Osbourn AE (2003) Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus. Plant Mol Biol 51:731–743
Jin ML, Lee DY, Um Y, Lee JH, Park CG, Jetter R, Kim OT (2014) Isolation and characterization of an oxidosqualene cyclase gene encoding a β-amyrin synthase involved in Polygala tenuifolia Willd. Saponin biosynthesis. Plant Cell Rep 33:511–519
Kawano N, Ichinose K, Ebizuka Y (2002) Molecular cloning and functional expression of cDNAs encoding oxidosqualene cyclases from Costus speciosus. Biol Pharm Bull 25:477–482
Kemen AC, Honkanen S, Melton RE, Findlay KC, Mugford ST, Hayashi K, Haralampidis K, Rosser SJ, Osbourn A (2014) Investigation of triterpene synthesis and regulation in oats reveals a role for β-amyrin in determining root epidermal cell patterning. Proc Natl Acad Sci USA 23:8679–8684
Kim YS, Cho JH, Ahn J, Hwang B (2006) Upregulation of isoprenoid pathway genes during enhanced saikosaponin biosynthesis in the hairy roots of Bupleurum falcatum. Mol Cells 22:269–274
Kim YS, Cho JH, Park S, Han JY, Back K, Choi YE (2011) Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum. Planta 233:343–355
Kushiro T, Shibuya M, Ebizuka Y (1998) β-Amyrin synthase: cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur J Biochem 256:238–244
Law BYK, Mo JF, Wong VKW (2014) Autophagic effects of chaihu (dried roots of Bupleurum chinense DC. or Bupleurum scorzoneraefolium Willd.). Chin Med 9:21
Lin TY, Chiou CY, Chiou SJ (2013) Putative genes involved in saikosaponin biosynthesis in Bupleurum species. Int J Mol Sci 14:12806–12826
Liu XL (2010) Molecular cloning and expression pattern of key enzyme genes in saikosaponin biosynthesis pathway in Bupleurum chinense DC. Post-doctoral dissertation supervised by Chen SL & Wei JH in IMPLAD, Beijing
Liu WY, Peng PH, Lin TY (2006) Cloning and characterization of beta-amyrin synthase from Bupleurum kaoi. 8th international congress of plant molecular biology. Book of abstracts, ISPMB, Adelaide, 20–25 Aug 2006, POS-TUE-121
Liu YL, Cai YF, Zhao ZJ, Wang JF, Li J, Xin W, Xia GM, Xiang FN (2009) Cloning and functional analysis of a β-amyrin synthase gene associated with oleanolic acid biosynthesis in Gentiana straminea Maxim. Biol Pharm Bull 32:818–824
Mabberley DJ (2008) Mabberley’s plant-book: a portable dictionary of plants, their classification and uses, 3rd edn. Cambridge University Press, New York
Morita M, Shibuya M, Kushiro T, Masuda K, Ebizuka Y (2000) Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum) new alpha-amyrin-producing enzyme is a multifunctional triterpene synthase. Eur J Biochem 267:3453–3460
Moses T, Pollier J, Almagro L, Buyst D, Van Montagu M, Pedreño MA, Martins JC, Thevelein JM, Goossens A (2014) Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum. Proc Natl Acad Sci USA 111:1634–1639
Pan SL (2006) Bupleurum species: scientific evaluation and clinical applications. CRC/Taylor & Francis, Boca Raton
Patra B, Schluttenhofer C, Wu Y, Pattanaik S, Yuan L (2013) Transcriptional regulation of secondary metabolite biosynthesis in plants. Biochim Biophys Acta 1829:1236–1247
Sawai S, Shindo T, Sato S, Kaneko T, Tabata S, Ayabe SI, Aoki T (2006) Functional and structural analysis of genes encoding oxidosqualene cyclases of Lotus japonicus. Plant Sci 170:247–257
Shan RH, She ML (1979) Flora of China, vol 55. Science Press, Beijing, pp 215–295
Shibuya M, Katsube Y, Otsuka M, Zhang H, Tansakul P, Xiang T, Ebizuka Y (2009) Identification of a product specific beta-amyrin synthase from Arabidopsis thaliana. Plant Physiol Biochem 47:26–30
Staniek A, Bouwmeester H, Fraser PD, Kayser O, Martens S, Tissier A, van der Krol S, Wessjohann L, Warzecha H (2013) Natural products—modifying metabolite pathways in plants. Biotechnol J 8:1159–1171
Sui C, Zhang J, Wei JH, Chen SL, Li Y, Xu JS, Jin Y, Xie CX, Gao ZH, Chen HL, Yang CM, Zhang Z, Xu YH (2011) Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins. BMC Genomics 12:539
Takagi K, Nishizawa K, Hirose A, Kita A, Ishimoto M (2011) Manipulation of saponin biosynthesis by RNA interference-mediated silencing of β-amyrin synthase gene expression in soybean. Plant Cell Rep 30:1835–1846
Thimmappa R, Geyser K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:16.1–16.33
Van Wyk BE, Wink M (2004) Medicinal plants of the world: an illustrated scientific guide to important medicinal plants and their uses. Timber Press, Portland
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Gao, K., Wu, Sr., Wang, L. et al. Cloning and analysis of β-amyrin synthase gene in Bupleurum chinense . Genes Genom 37, 767–774 (2015). https://doi.org/10.1007/s13258-015-0307-0
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DOI: https://doi.org/10.1007/s13258-015-0307-0