Plant Growth Regul (2013) 71:207–217 DOI 10.1007/s10725-013-9821-8 ORIGINAL PAPER Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences Yao Sun • Shou-Jing Zhao • Yan-Long Liang • Wang le • Hao-Jie Cao Received: 6 December 2012 / Accepted: 30 April 2013 / Published online: 11 May 2013 Ó The Author(s) 2013. This article is published with open access at Springerlink.com Abstract Asian ginseng (Panax ginseng) and American medium. In vivo, we established both PgCYP716A47 and ginseng (Panax quinquefolium), are thought to be repre- PqD12H RNAi transgenic. The RT-PCR and HPLC anal- sentative plant of Panax species, have important com- ysis of the final products of protopanaxadiol and proto- mercial value and are used in worldwide. Panax species panaxatriol showed a result that declined level of produces triterpene saponins called ginsenosides, which are protopanaxadiol-type and protopanaxatriol-type ginseno- classified into two groups by the skeleton of aglycones, sides. It suggested that the P450 synthase content or namely dammarane-type and oleanane-type. Dammarane- expression in American ginseng exceed than in Asian type ginsenosides dominate over oleanane-type not only in ginseng. The result elucidated the evolution relationship of amount but also in structural varieties. Researches shows P450s and the reason of different saponins content among that the saponins content in American ginseng is higher Panax species. than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed Keywords Panax ginseng Á Panax quinquefolius Á that protopanaxadiol derived from dammarenediol-II, is a Protopanaxdiol Á P450 Á RNA interference key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng Introduction (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American Asian ginseng (Panax ginseng) and American ginseng ginseng ginsenoside biosynthesis. Here, we first cloned and (Panax quinquefolius) are the two most commonly used identified a P450 gene named PqD12H encoding enzymes ginseng herbs and are thought to have exceptional curative catalyzed dammarenediol-II to protopanaxadiol by RT- properties, have an important commercial value and are PCR using degenerate primers designed based on sequence used worldwide (Kitts et al. 2000). Although American homology. In vitro, the ectopic expression of PqD12H in ginseng is native to North American, it has spread to North recombinant WAT21 yeast resulted in protopanaxadiol Asian (Assinewe et al. 2003). Ginsenosides, which are production after dammarenediol-II was added to the culture glycosylated triterpenes, are considered to be the main compounds in American ginseng, showing many pharma- cological activities including anti-cancer, anti-diabetic, Yan-Long Liang, Wang le, Hao-Jie Cao assisted to this work. neuroprotective, radioprotective, anti-amnestic and anti- aging effects (Chen et al. 2011). Both Asian and American Y. Sun Á S.-J. Zhao (&) Á Y.-L. Liang Á W. le Á H.-J. Cao ginseng contained a group of saponins generally referred to School of Biological and Agricultural Engineering, Jilin as ginsenosides. Up till now, saponins including ginseno- University, No. 5988 Renmin Street, Nanguan District, Changchun, People’s Republic of China sides Rg1, Re, Rb1, Rg2, Rb2, Rc and Rd, have been e-mail: [email protected] widely recognized as the main active ingredients of both Y. Sun the two plants (Devi et al. 2011; Barton et al. 2010). e-mail: [email protected] However, American ginseng has been used as a tonic for 123 208 Plant Growth Regul (2013) 71:207–217 indications similar to those of Asian ginseng, but the two ginseng, based on homologous sequence analysis and species differ in their respective quantities of the specific amplified by RT-PCR. We constructed both PgCYP716 ginsenosides (Lee et al. 2008; Schlag and McIntosh 2006; A47 and PqD12H RNAi destination vector based on Chen et al. 2009). recombinant PCR from Asian and American ginseng The current ideas of ginsenoside biosynthesis (Fig. 1), 4 year roots. Functionally analyzed by ectopic expression considered ginsenosides to be derived from the 2.3-ox- of PqD12H in yeast yielded protopanaxadiol and mea- idosqualene, which can be synthesized from the plastidial sured of protopanaxadiol and protopanaxatriol products mevalonic acid pathway (MVA) (Augustin et al. 2011; activity from RNAi transgenic, indicating that PqD12H is Liang and Zhao 2008). From 2.3-oxidosqualene, the pri- protopanaxadiol synthase, which is a critically important mary components of triterpenoid saponins are oleanane step in ginsenoside biosynthesis. RNAi in transgenic hairy (b-amyrin), cycloartenol, lupeol or dammarene-type tri- roots also showed that even though the protopanaxadiol terpenoid. Ginsenosides are classified into two groups by synthase was inhibited, P450 enzyme content or expres- the structure of aglycones, dammarane-type and oleanane- sion level in American ginseng is exceed than in Asian type. Major ginsenosides are dammarane-type, including ginseng. The objective of the work was to explain the Rb1 and Rg1 whose genuine aglycones are protopanaxa- reason of different to American and Asian ginseng in their diol and protopanaxatriol respectively (Punja 2012). respective quantities of the specific ginsenosides and Hydroxylation of dammarenediol-II by a P450 enzyme support the role of P450 in triterpenes saponins (D12H) at C-12 position generates protopanaxadiol then biosynthesis. direct hydroxylation of protopanaxadiol to protopanaxatriol involve in another P450 enzyme (P6H) at C-6 position (Cai et al. 2007). Dammarenediol-II is thought to be converted Results to a ginsenoside after hydroxylation by P450 enzymes and subsequent glycosylation by glycosyltransferase (GT). Isolation and analysis of PqD12H Both P450 and GT are in plant genome supergene families (Inoue 2005; Qi et al. 2011). Currently, substantial progress RT-PCR was performed with the designed degenerate has been obtained in dissecting the ginsenoside biosyn- primers, and several sequences were amplified. PCR thesis pathway; the genes encoding b-amyrin synthase, products were inserted into plasmid vector and sequenced. dammarenediol synthase have been cloned and identified An online BLAST P search revealed that one deduced from both the P. ginseng and P. quinquefolius (Han et al. amino acid sequence of these PCR products is most 2011). However, only the protopanaxadiol P450 has been possibly involved in ginsenoside biosynthesis due to its cloned from Asian ginseng (Han et al. 2012), for American high sequence similarity (99 and 98 %) with the cyto- ginseng there are few reports on cloning and identification chrome P450 (PgCYP716A47 and PnCYP450) catalyzing of genes encoding enzymes involved in dammarene-type the formation of protopanaxadiol from dammarenediol-II ginsenoside biosynthesis. in P. ginseng and P. notoginseng respectively (Fig. 2). The genes coding for enzymes of biochemical pathways Thus this sequence was selected for further studies and involved in triterpene biosynthesis are of considerable named as PqD12H. The cDNA of PqD12H (GeneBank interest in the area of ginseng biotechnology. Up regulation accession number JX569336) was 1,459-bp long, with a or down regulation of phytosterol and triterpene production 50-untranslated region of 10 nucleotides, a predicted open by genetic engineering of Panax species is an attractive reading frame (ORF) of 1,449 nucleotides encoding a strategy to achieve a higher medicinal value (Yue and protein of 482 amino acids with a calculated molecular Zhong 2005). A more detailed understanding of genes mass of 60 kDa. According to the P450 nomenclature and involved in saponin biosynthesis could facilitate the the results of BLAST P, PqD12H should belong to genetic modification of plants with altered or novel saponin CYP716A subfamily involved in terpenoid metabolism content (Nelson 2006). Plant functional genomics involves (Devi et al. 2011; Han et al. 2011). To further evaluate generation of transgenic and mutant plants in association the homology and evolution relationship of PqD12H with with multiparallel analysis of gene products such as mRNA other P450 genes from Panax species and other various and protein. In the last few years, significant technological species, phylogenetic tree was constructed (Fig. 3)at progress has been made that will facilitate the generation amino acid level. As expected, it could be observed that and characterization of genetic diversity in plant systems PqD12H is most closely related to PgCYP716A47 and (Li et al. 2009). PnCYP450 from P. ginseng and P. notoginseng.This In this study, we first cloned a P450 gene (PqD12H) indicated that PqD12H is most probably a gene encoding encoding enzymes catalyzing the reaction from damma- enzyme catalyzing the reaction from dammarenediol-II to renediol-II to protopanaxadiol from 4 years American protopanaxadiol. 123 Plant Growth Regul (2013) 71:207–217 209 Fig. 1 The proposed biosynthetic pathway of ginsenosides. MVA mevalonate, IPP isopentenyl diphosphate, GPP geranyl diphosphate, FPP farnesyl diphosphate, GPS geranyl diphosphate synthase, FPS farnesyl diphosphate synthase, SS squalene synthase, SE squalene epoxidase, CAS cycloartenol synthase, LS lupeol synthase, BAS beta-amyrin synthase, DS dammarenediol