De Novo RNA Sequencing and Expression Analysis of Aconitum carmichaelii to Analyze Key Genes Involved in the Biosynthesis of Diterpene Alkaloids

Abstract

Aconitum carmichaelii is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of A. carmichaelii has shown accumulation of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root) of A. carmichaelii, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of A. carmichaelii with A. heterophyllum identified 20,232 orthogroups, representing 30,633 unigenes of A. carmichaelii, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.

Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

“…Gene expansion followed by neo/sub‐functionalization plays an important role in the diversification of secondary metabolites (Rai et al ., 2017a,b). Therefore, we evaluated the gene expansion of candidate genes assigned to emerged cadaverine catabolic pathways across nine plant species: three non‐cadaverine and non‐functional‐ODC plants ( A. thaliana , Brassica rapa , and Physcomitrella patens ); two non‐cadaverine and functional‐ODC plants ( Malus domestica and Papaver somniferum ); and four cadaverine and functional‐L/ODC plants ( Glycine max , L. angustifolius , Medicago truncatula and Nelumbo nucifera ).…”

“…Accession IDs for genome assemblies of each plant are provided in Table S8. Orthologs of candidate genes were obtained using orthofinder 2.31, as described previously (Rai et al ., ; Sun et al ., ). Gene gain, loss, expansion and contraction in each orthogroup were evaluated in the count package, as noted previously (Li et al ., ).…”

“…As a rough estimate the plant kingdom synthesizes over 1 000 000 metabolites with diverse physiological effects, and several of these have also proven to possess beneficial properties for humans (Saito and Matsuda, ; Afendi et al ., ; Yamazaki et al ., ). Since ancient times, therefore, plant metabolite extracts have been used as medicines and as lead compounds to inspire new drug molecules with various pharmacological properties (Kinghorn et al ., ; Rai et al ., 2017a,b).…”

Natural Voltage‐Gated Sodium Channel Ligands: Biosynthesis and Biology

“…GGPP is cyclized by ent ‐copalyl diphosphate synthase to form ent ‐copalyl diphosphate ( 35 ) which undergoes a rearrangement to form 33 , similar to the proposed pathway for grayanotoxins . Candidate genes for enzymes responsible for the diversification of the aconitine scaffold have been identified through transcriptome analysis of Aconitum carmichaelii flowers, buds, leaves, and roots, revealing the possible involvement of cytochrome P450s, methyltransferases, and acyltransferases . Zhao and co‐workers proposed a putative biosynthetic pathway based on the proposed function of each gene product assigned through BLAST analysis .…”

“…[66] Candidate genes fore nzymes responsible for the diversification of the aconitines caffold have been identified through transcriptome analysis of Aconitumc armichaelii flowers, buds, leaves,a nd roots, revealing the possible involve- ChemBioChem 2019ChemBioChem , 20,1231ChemBioChem -1241 www.chembiochem.org ment of cytochrome P450s, methyltransferases, and acyltransferases. [66,67] Zhao and co-workers proposed ap utative biosynthetic pathway based on the proposed functiono fe ach gene product assignedt hrough BLAST analysis. [66] It is proposed that 33 is furtherm odified by cyclases capable of as imultaneous ring expansion and contraction form the putative core intermediate (36).…”