Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis

Abstract

Background: Eleutherococcus senticosus, Siberian ginseng, is a highly valued woody medicinal plant belonging to the family Araliaceae. E. senticosus produces a rich variety of saponins such as oleanane-type, noroleanane-type, 29-hydroxyoleanan-type, and lupane-type saponins. Genomic or transcriptomic approaches have not been used to investigate the saponin biosynthetic pathway in this plant.

Result: In this study, de novo sequencing was performed to select candidate genes involved in the saponin biosynthetic pathway. A half-plate 454 pyrosequencing run produced 627,923 high-quality reads with an average sequence length of 422 bases. De novo assembly generated 72,811 unique sequences, including 15,217 contigs and 57,594 singletons. Approximately 48,300 (66.3%) unique sequences were annotated using BLAST similarity searches. All of the mevalonate pathway genes for saponin biosynthesis starting from acetyl-CoA were isolated. Moreover, 206 reads of cytochrome P450 (CYP) and 145 reads of uridine diphosphate glycosyltransferase (UGT) sequences were isolated. Based on methyl jasmonate (MeJA) treatment and real-time PCR (qPCR) analysis, 3 CYPs and 3 UGTs were finally selected as candidate genes involved in the saponin biosynthetic pathway.

Conclusions: The identified sequences associated with saponin biosynthesis will facilitate the study of the functional genomics of saponin biosynthesis and genetic engineering of E. senticosus.

Figure 1

Length distribution of the assembled contigs of E. senticosus .

Figure 2

Histogram presentation of functional annotations of the unique sequences by the gene ontology classification. The results are summarised in three main categories: cellular component, molecular function and biological process. The right y-axis indicates the number of genes in a category. The left y-axis indicates the percentage of unique sequences in a specific category.

Figure 3

Putative saponin biosynthetic pathway from 2,3-oxidosquane in E. senticosus .

Figure 4

Phylogenetic tree of the deduced amino acid sequences of EsBAS and other plant OSCs. Phylogenetic trees of plant OSC distances between each clone and group were calculated using the program CLUSTAL W. The distance between each clone was calculated using CLUSTAL W. Bootstrap analysis values are shown at the nodal branches. The indicated scale represents 0.1 amino acid substitutions per site. Pg, Panax ginseng; Aa, Artemisia annua; Es, Eleutherococcus senticosus; Bp, Betula platyphylla; Et, Euphorbia tirucalli; Vh, Vaccaria hispanica; Lj, Lotus japonicas; Gg, Glycyrrhiza glabra; Ps, Pisum sativum; Mt, Medicago truncatula; At, Arabidopsis thaliana; Bg, Bruguiera gymnorrhiza; Pv, Panax vietnamensis; Oe, Olea europaea; To, Taraxacum officinale; Cs, Crocus speciosus; Ca, Centella asiatica.

Figure 5

qPCR analysis of 22 CYPs and EsBAS in MeJA-treated leaves of E. senticosus . The relative fold expression of genes in MeJA-treated leaves and untreated controls is shown. EsBAS, putative β-amyrin synthase in E. senticosus.

Figure 6

Phylogenetic tree of the deduced amino acid sequences of EsCYP-03, 17, 18 and other plant CYPs. Phylogenetic trees of plant OSC distances between each clone and group were calculated using the program CLUSTAL W. The distance between each clone was calculated using CLUSTAL W. Bootstrap analysis values are shown at the nodal branches. The indicated scale represents 0.1 amino acid substitutions per site. Pg, Panax ginseng; Es, Eleutherococcus senticosus; Cr, Catharanthus roseus; Mt, Medicago truncatula; Vv, Vitis vinifera; Gu, Glycyrrhiza uralensis; As, Avena strigose; At, Arabidopsis thaliana; Gm, Glycine max.

Figure 7

qPCR analysis of 15 selected UGTs of E. senticosus in MeJA-treated materials.

Figure 8

Phylogenetic tree of the deduced amino acid sequences of EsUGT-3, 10, 11 and other plant UGTs. Phylogenetic trees of plant OSC distances between each clone and group were calculated using the program CLUSTAL W. The distance between each clone was calculated using CLUSTAL W. Bootstrap analysis values are shown at the nodal branches. The indicated scale represents 0.1 amino acid substitutions per site. Bv, Barbarea vulgaris; Es, Eleutherococcus senticosus; Gm, Glycine max; Mt, Medicago truncatula; Vh, Vaccaria hispanica; At, Arabidopsis thaliana.