Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

Abstract

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium), are thought to be representative plant of Panax species, have important commercial value and are used in worldwide. Panax species produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Dammaranetype ginsenosides dominate over oleanane-type not only in amount but also in structural varieties. Researches shows that the saponins content in American ginseng is higher than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed that protopanaxadiol derived from dammarenediol-II, is a key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American ginseng ginsenoside biosynthesis. Here, we first cloned and identified a P450 gene named PqD12H encoding enzymes catalyzed dammarenediol-II to protopanaxadiol by RT-PCR using degenerate primers designed based on sequence homology. In vitro, the ectopic expression of PqD12H in recombinant WAT21 yeast resulted in protopanaxadiol production after dammarenediol-II was added to the culture medium. In vivo, we established both PgCYP716A47 and PqD12H RNAi transgenic. The RT-PCR and HPLC analysis of the final products of protopanaxadiol and protopanaxatriol showed a result that declined level of protopanaxadiol-type and protopanaxatriol-type ginsenosides. It suggested that the P450 synthase content or expression in American ginseng exceed than in Asian ginseng. The result elucidated the evolution relationship of P450s and the reason of different saponins content among Panax species.

Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius

“…In the present study, we isolated and regulated a (Kim et al, 2014;Park et al, 2016;Rahimi et al, 2015;Wang et al, 2014b;Zhao et al, 2015). Moreover, RNAi has been used for the identification or validation of biological function of the targeted transcripts of individual genes in planta (Han et al, 2010;Sun et al, 2013). Many reports indicated the possibility of altering ginsenoside levels by regulating genes encoding enzymes involved in ginsenoside biosynthesis.…”

“…It has reported that inhibiting the expression of protopanaxadiol synthase can depress the levels of protopanaxadiol and protopanaxatriol. Moreover, they also suggested that the P450 synthase content in P. quinquefolius is exceeded than that in P. ginseng (Sun et al, 2013). The RNAi-mediated inhibition of β-Amyrin synthase decreased levels of oleanane-type ginsenoside Ro and increased the level of total ginsenosides in P. ginseng (Zhao et al, 2015).…”

“…The PCR product was purified, ligated to pGEM-T vector, sequenced, excised and re-cloned between the Sam І and Sac І of pAUR123 expression vector (TaKaRa Dalian China). The recombinant expression vector and an empty vector were transformed into Saccharomyces cerevisiae strain INVSc1 yeast according to the previous studies with some modifications (Sun et al, 2013;Wang et al, 2014b).Transformed cells were selected using antibiotics aureobasidin A (AbA) after 4 days at 30 °C and then subcultured on YPD liquid medium.…”

“…Ginsenosides of both transgenic hairy root lines and control groups of P. ginseng and P. quinquefolius were extracted and analyzed high-performance liquid chromatography (HPLC) according to the protocol reported in our lab (Sun et al, 2013;Zhao et al, 2015). The HPLC separation was performed on a C18 column (5 μm, 4.6×250 mm, Shimadzu, Kyoto, Japan), maintained at 30 °C, with water and acetonitrile as mobile phase.…”

“…To functionally analyze the role of two UGT genes in transgenic P. ginseng and P.quinquefolius plants, constitutively expressed 3-O-UGT2-RNAi component was generated according to a highly conserved nucleotide domain within two genes. The induction and selection of all transgenic root lines were performed using the protocol described in our previous reports (Sun et al, 2013;Zhao et al, 2015).The hairy root originated from independent root discs of P. quinquefolius and P. ginseng was considered as an independent sample. After being subcultured for more than five generations in liquid MS medium, the control and RNAi root lines were used for further analysis.…”