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

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. Dammarane-type 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.