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. 2016 Apr 12;7:481.
doi: 10.3389/fpls.2016.00481. eCollection 2016.

RNA-seq Transcriptome Analysis of Panax Japonicus, and Its Comparison With Other Panax Species to Identify Potential Genes Involved in the Saponins Biosynthesis

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Free PMC article

RNA-seq Transcriptome Analysis of Panax Japonicus, and Its Comparison With Other Panax Species to Identify Potential Genes Involved in the Saponins Biosynthesis

Amit Rai et al. Front Plant Sci. .
Free PMC article

Abstract

The Panax genus has been a source of natural medicine, benefitting human health over the ages, among which the Panax japonicus represents an important species. Our understanding of several key pathways and enzymes involved in the biosynthesis of ginsenosides, a pharmacologically active class of metabolites and a major chemical constituents of the rhizome extracts from the Panax species, are limited. Limited genomic information, and lack of studies on comparative transcriptomics across the Panax species have restricted our understanding of the biosynthetic mechanisms of these and many other important classes of phytochemicals. Herein, we describe Illumina based RNA sequencing analysis to characterize the transcriptome and expression profiles of genes expressed in the five tissues of P. japonicus, and its comparison with other Panax species. RNA sequencing and de novo transcriptome assembly for P. japonicus resulted in a total of 135,235 unigenes with 78,794 (58.24%) unigenes being annotated using NCBI-nr database. Transcriptome profiling, and gene ontology enrichment analysis for five tissues of P. japonicus showed that although overall processes were evenly conserved across all tissues. However, each tissue was characterized by several unique unigenes with the leaves showing the most unique unigenes among the tissues studied. A comparative analysis of the P. japonicus transcriptome assembly with publically available transcripts from other Panax species, namely, P. ginseng, P. notoginseng, and P. quinquefolius also displayed high sequence similarity across all Panax species, with P. japonicus showing highest similarity with P. ginseng. Annotation of P. japonicus transcriptome resulted in the identification of putative genes encoding all enzymes from the triterpene backbone biosynthetic pathways, and identified 24 and 48 unigenes annotated as cytochrome P450 (CYP) and glycosyltransferases (GT), respectively. These CYPs and GTs annotated unigenes were conserved across all Panax species and co-expressed with other the transcripts involved in the triterpenoid backbone biosynthesis pathways. Unigenes identified in this study represent strong candidates for being involved in the triterpenoid saponins biosynthesis, and can serve as a basis for future validation studies.

Keywords: Panax japonicas; RNA-seq; comparative transcriptomics analysis; cytochrome P450; triterpenoid saponins.

Figures

FIGURE 1
FIGURE 1
Five tissues of P. japonicus used for transcriptome study. Five tissues of P. japonicus, namely, leaf (a), flower (b), rhizome_Y (c), rhizome_O (d), and secRoot (e) were used to performed transcriptome profiling, and de novo transcriptome assembly. Scale bars for each panel measure to 0.5 cm.
FIGURE 2
FIGURE 2
Overview of the de novo transcriptome assembly in P. japonicus. (A,B) represents length and GC distribution of contigs assembled from all cleaned reads from five tissues of P. japonicus using Trinity program (Grabherr et al., 2011), (C,D) represents length and GC distribution of unigenes generated from further contigs assembly.
FIGURE 3
FIGURE 3
Characterization of P. japonicus unigenes based on NCBI non-redundant (nr) protein database search. (A) E-value distribution of Blast hits for the P. japonicus assembled transcriptome with a cutoff of E-value < 10-5. (B) Similarity score distribution plot of top Blast hits for the assembled unigenes. (C) Bar chart of the data distribution based on blast search and annotation using Blast2GO. (D) Species distribution of the top Blast hits for the assembled unigenes.
FIGURE 4
FIGURE 4
Gene ontology (GO) annotation for all assembled unigenes in P. japonicus. GO-terms for all unigenes were assigned based on Blast search results using Blast2GO program at GO annotation level 5. The results are summarized in terms of three functional categories, namely, biological process, molecular function, and cellular component.
FIGURE 5
FIGURE 5
Distribution of unigenes and gene ontology enrichment analysis for five tissues of P. japonicus. (A) Venn diagram for unigenes with non-zero FPKM values in individual tissues. (B–F) Gene ontology enrichment analysis using Fisher’s exact test with P-value cutoff applied as 0.05. Unigenes for each tissue with FPKM value above 10 were selected as test set, and used against the P. japonicus transcriptome assembly as reference set to identify differential GO-terms enriched in the specific tissue, with P-value being calculated based on reference set.
FIGURE 6
FIGURE 6
Putative triterpenoid saponins biosynthesis pathway and expression level of associated unigenes across five tissues in P. japonicus. (A) Proposed pathway for triterpenoid saponins biosynthesis. (B) Expression levels of the candidate unigenes that showed high sequence similarity with other Panax species, and were annotated as enzyme coding genes from triterpenoid backbone biosynthesis pathways. The expression value (FPKM) for unigenes across all tissues were log2 transformed and scaled across each row, and heatmap was generated using R-package heatmap2.0. AACT, Acetyl-CoA acetyltransferase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MVK, mevalonate kinase; PMK, phosphor mevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IPI, isopentenyl diphosphate isomerase; GPPS, geranylgeranyl pyrophosphate synthase; FPS, farnesyl diphosphate synthase; SS, squalene synthase; SE, squalene epoxidase; DS, dammarendiol synthase; AS, beta-amyrin synthase; GTs, UDP glycosyltransferase; P450, cytochrome P450; FPKM, fragments per kilobase per million.
FIGURE 7
FIGURE 7
K-mean clustering for unigenes annotated as CYP450s or associated with sugar conjugation processes together with unigenes from triterpenoid backbone biosynthetic pathways. Unigenes annotated as cytochrome P450, or sugar conjugation associated processes were selected from the P. japonicus transcriptome assembly, resulting in total of 870 unigenes. These gene groups were used together with 28 unigenes annotated as enzyme coding genes from triterpenoid backbone biosynthetic pathways, and k-mean clustering analysis was performed based on gene expression values across all five tissues of P. japonicus. Distance matrix for k-mean clustering was calculated by Euclidean similarity measurement with 10,000 iterations, resulting in 6 gene clusters. Name of unigenes associated with triterpenoid backbone biosynthetic pathways and identified in a particular cluster is represented within each gene group.
FIGURE 8
FIGURE 8
Expression profile for putative cytochrome P450 (CYP450s) and GTs unigenes from ginsenosides biosynthetic pathways across five tissues of P. japonicus. The expression value (FPKM) for unigenes, annotated as CYP450 or GTs and co-expressed with triterpenoid saponin biosynthetic pathways, were log2 transformed and scaled across each row, and heatmap was generated using R-package heatmap2.0.
FIGURE 9
FIGURE 9
Phylogenetic analysis of putative CYP450s unigenes from P. japonicus transcriptome with all CYP450 genes from Arabidopsis genome. Protein sequences were aligned using MUSCLE program, and evolutionary distances were computed using Jones-Taylor-Thornton (JTT) method. A Neighbor-Joining (NJ) tree was constructed with bootstrap values obtained after 10,000 replications using MEGA6 program.
FIGURE 10
FIGURE 10
Phylogenetic analysis of putative GTs unigenes from P. japonicus transcriptome with 128 genes annotated as GTs from Arabidopsis genome. Protein sequences were aligned using MUSCLE program, and evolutionary distances were computed using JTT method. A NJ tree was constructed with bootstrap values obtained after 10,000 replications using MEGA6 program.

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