De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis

Abstract

Background: American ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway.

Results: In this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8%) unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450) and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA) inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay.

Conclusions: We demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community that is interested in the molecular genetics and functional genomics of American ginseng.

Figure 1

Putative ginsenoside biosynthetic pathway in American ginseng. AACT, acetyl-CoA acetyltransferase; AS, β-amyrin synthase; CAS, cycloartenol synthase; DS, dammarenediol-II synthase; FPS, farnesyl diphosphate synthase; GT, glycosyltransferase; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IDI, isopentenyl diphosphate isomerase; MDD, mevalonate diphosphate decarboxylase; MK, mevalonate kinase; P450, cytochrome P450; PMK, phosphomevalonate kinase; Rb-1, ginsenoside Rb-1; Rg-1, ginsenoside Rg-1; Ro, ginsenoside Ro; SQE, squalene epoxidase; and SQS, squalene synthase.

Figure 2

Size distribution of the 454 HQ reads and the contigs assembled from them. A) 454 HQ reads; B) contigs.

Figure 3

Expression patterns of OSCs in different plant tissues. A) OSC expression in the root, stem, leaf, and flower of American ginseng. B) Comparison of the relative abundance of OSC transcripts in American ginseng roots resulting from real-time PCR and EST counting, respectively.

Figure 4

Real time PCR analysis of CYP450s in MeJA-treated materials and different plant tissues. CK represents the uninduced material; 6H represents the material treated for 6 h with MeJA; and DS represents dammarenediol-II synthase. The corresponding contigs represented by numbers 1 - 27 are listed in Additional File 6. A) Changes in the gene expression of CYP450s induced by MeJA. B) The gene expression of CYP450s in different tissues.

Figure 5

Real-time PCR analysis of UGTs in MeJA-treated materials and different plant tissues. R represents root; S represents stem; L represents leaf; F represents flower and DS represents dammarenediol-II synthase. The corresponding contigs represented by numbers 1 - 27 are listed in Additional File 6. A) Changes in gene expression of UGTs induced by MeJA. B) The gene expression of UGTs in different tissues.