Comparative transcriptome analysis of root, stem, and leaf tissues of Entada phaseoloides reveals potential genes involved in triterpenoid saponin biosynthesis

Abstract

Background: Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant.

Results: We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR.

Conclusions: The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of Entada phaseoloides.

Keywords: Entada phaseoloides; Secondary metabolites; Transcriptome; Triterpenoid saponins.

Fig. 1

Similarity of unigenes annotated using the Nr database. a E-value distribution of best BLAST hits for each unigene (E-value <1e-5). b Similarity distribution of top BLAST hits for each unigene. c Distribution of the most homologous sequence results for each unigene by species (E-value <1e-5)

Fig. 2

KEGG pathway classification of Entada phaseoloides unigenes. Exactly 15,119 unigenes were divided into five main categories in accordance with the corresponding pathways

Fig. 3

Differential expression analysis of unigenes. a Number of DEGs in each tissue compared with the other two tissues; b Venn diagram representing the number of DEGs among Entada phaseoloides tissues

Fig. 4

KEGG enrichment analyses of DEGs in different tissues. a between leaf and stem; b between leaf and root; c between stem and root

Fig. 5

Schematic representation of the potential triterpenoid saponin biosynthesis pathway. Transcriptomic data (lg FPKM) for each gene represent the expression in the root (R), stem (S), and leaf (L) on heat map

Fig. 6

Phylogenetic analysis of the EpBAS and other plant BASs. The distances between each clone and group were calculated using CLUSTAL W. Bootstrap values are shown. Ap: Abrus precatorius; Gm: Glycine max; Gi: Glycyrrhiza inflata; Gu: Glycyrrhiza uralensis; Ep: Entada phaseoloides; Mt: Medicago truncatula; Lj: Lotus japonicus; Pt: Polygala tenuifolia; Pp: Prunus persica

Fig. 7

Heat map representing the upregulated unigenes of CYP450s (a) and UGTs (b) in stem (S) compared with the root (R) and leaf (L). The fold change expression data were obtained after three biological replicates

Fig. 8

qRT-PCR validation of selected genes related to triterpene saponin biosynthesis. AACT: acetyl-CoA acetyltransferase; HMGS: hydroxymethyl- glutaryl-CoA synthase; HMGR: hydroxymethylglutaryl-CoA reductase; MK, mevalonate kinase; PMK: Phosphomevalonate kinase; IPI: isopentenyl pyrophosphate isomerase; FPS: farnesyl pyrophosphate synthase; SS: squalene synthase; SE: squalene epoxidase