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Planta Med 2018; 84(12/13): 920-934
DOI: 10.1055/a-0630-5925
Natural Product Chemistry and Analytical Studies
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

De Novo Transcriptome Assembly and Characterization of Lithospermum officinale to Discover Putative Genes Involved in Specialized Metabolites Biosynthesis[*]

Amit Rai
1   Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
,
Taiki Nakaya
1   Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
,
Yohei Shimizu
1   Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
,
Megha Rai
1   Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
,
Michimi Nakamura
1   Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
,
Hideyuki Suzuki
2   Kazusa DNA Research Institute, Chiba, Japan
,
Kazuki Saito
1   Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
,
Mami Yamazaki
1   Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
› Author Affiliations
Further Information

Publication History

received 07 March 2018
revised 06 May 2018

accepted 08 May 2018

Publication Date:
29 May 2018 (online)

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Abstract

Lithospermum officinale is a valuable source of bioactive metabolites with medicinal and industrial values. However, little is known about genes involved in the biosynthesis of these metabolites, primarily due to the lack of genome or transcriptome resources. This study presents the first effort to establish and characterize de novo transcriptome assembly resource for L. officinale and expression analysis for three of its tissues, namely leaf, stem, and root. Using over 4Gbps of RNA-sequencing datasets, we obtained de novo transcriptome assembly of L. officinale, consisting of 77,047 unigenes with assembly N50 value as 1524 bps. Based on transcriptome annotation and functional classification, 52,766 unigenes were assigned with putative genes functions, gene ontology terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene ontology enrichment analysis using highly expressed unigenes across three tissues and targeted metabolome analysis showed active secondary metabolic processes enriched specifically in the root of L. officinale. Using co-expression analysis, we also identified 20 and 48 unigenes representing different enzymes of lithospermic/chlorogenic acid and shikonin biosynthesis pathways, respectively. We further identified 15 candidate unigenes annotated as cytochrome P450 with the highest expression in the root of L. officinale as novel genes with a role in key biochemical reactions toward shikonin biosynthesis. Thus, through this study, we not only generated a high-quality genomic resource for L. officinale but also propose candidate genes to be involved in shikonin biosynthesis pathways for further functional characterization.

Key words

Lithospermum officinale - Boraginaceae - shikonin biosynthesis - chlorogenic acid - lithospermic acid - RNA-sequencing - deep transcriptome analysis - KEGG-pathways enrichment analysis

* Dedicated to Professor Dr. Robert Verpoorte in recognition of his outstanding contribution to natural products research.


Supporting Information

    Nucleotide sequences for unigenes of L. officinale, associated with lithospermic/chlorogenic acid and shikonin biosynthesis pathways, are provided as Supplementary Data 1 and Supplementary Data 2, respectively. Summary of L. officinale raw reads filtering using Trimmomatic program is presented in Table 1S. KEGG pathways identified in L. officinale transcriptome assembly is listed in Table 2S. Full accession list of CYPs used for phylogenetic analysis is in Table 3S. GC plot and length distribution plot of unigenes, InterProScan analysis, GO enrichment analysis across three tissues, lithospermic/chlorogenic acid, and shikonin biosynthesis co-expression analysis of L. officinale are shown in Fig. 1S–5S, respectively. Data on annotation and functional categories assigned to L. officinale unigenes are available at the authors on request. Further, all identified SSRs as well as associated unigenes of L. officinale are available at the authors on request.

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