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

Chao Sun,Hongmei Luo,Edmund Mk Lui,Qiong Wu,Ying Li,Yongzhen Sun,Jingyuan Song,Shilin Chen

doi:10.1186/1471-2164-11-262

Abstract

BackgroundAmerican 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.ResultsIn 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.ConclusionsWe 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.

Highlights

American ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world
The Roche 454 company prefers to resolve the polyA problem by cDNA synthesis primed with random hexamers, our study proved that enzyme digestion is at least an effective alternative way to eliminate polyA and is a promising method in 454 cDNA library preparation starting from total RNA
American ginseng is a suitable subject for the study of triterpene saponin biosynthesis

Summary

Introduction

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. Little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway. American ginseng (Panax quinquefolius L.) is a perennial understory herb from the Araliaceae family, which is native to the eastern forests of North America [1]. It is one of the most extensively used medicinal plants in both. The major bioactive components of American ginseng are the triterpene saponins known as ginsenosides.

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Conclusion