| Our group proposed the research platform on medicinal model organism, which should promote the modernization of traditional Chinese medicine (TCM). Salvia miltiorrhiza Bunge is one of the most commonly used medicinal plants in genus Salvia Linn, of Lamiaceae family, and it is highly valued for its dried roots and rhizomes called Danshen in TCM. S. miltiorrhiza is described as medicinal model plant in TCM research for its significant medicinal value, relatively small genome, short life cycles, efficient micropropagation and transgenic methods. The Danshen genome and transcriptome provide basis for elaborating the biosynthetic pathway and regulatory mechanism of active compounds. As the medicinal model plant, its functional genome still needs to be strengthened.This study focuses on the survey of full-length transcriptome by hybrid-seq (next generation and third generation sequencing) to identify alternative splicing events, investigate the consistency between gene expression and active compound contents, and predict the unknown biosynthetic encoding genes.1. Full-length transcriptome sequencing. This study firstly analyzed the full-length transcriptome in plant kingdom by hybrid-seq, and detected the alternative splicing events in S. miltiorrhiza.636,805 hybrid transcripts were produced with N50 of 2,411 bp. The length of hybrid transcripts was significantly improved in contrast to the assembly transcripts from only Illumina RNA-seq data with the N50 of 1,530 bp. The identification rate of full-length transcripts related to terpenoid biosynthesis from hybrid-seq was increased up to 71%. Based on Illumina short reads, hybrid reads, alternative splicing junctions, and genome data,4,035 isoforms and 14,241 isoforms were detected and predicted, respectively. Furthermore,40% multi-exon gene loci were identified as undergoing complex alternative splicing events.2. Identification of genes involved in tanshinone biosynthesis. The diterpenoid biosynthetic genes presented the highest expression in periderm of Danshen root, such as SmDXS2, SmDXR, SmHDS, SmHDRl, SmHDR3, SmIPIl, SmGGPPSl, SmCPS1, SmCPSS, SmKSL1 and SmKSL7 (Iog2 ratio≥ 1, FPKM> 10). The content of tanshinone IIA in periderm was 17 and 185 times higher than that in phloem and xylem, respectively. Here, we demonstrated that not only accumulation but also biosynthesis of the tanshinones occurs in the root periderm. Owing to co-expression with SmCPSl, SmKSLl and CYP76AH1,15 additional CYPs, one 2ODD, and five SDRs may play role(s) in tanshinone biosynthesis. In addition, candidate genes were cloned and used to construct RNAi and over-expression vectors, which were transformed into Agrobacterium rhizogenes to induce hairy roots of S. miltiorrhiza. Furthermore,20 full-length transcripts and 6 alternative splicing events were clearly observed among the genes involved in tanshinone biosynthesis, which may serve as a regulatory mechanism in controlling such diterpenoid metabolism.3. Identification of genes involved in salvianolic acid biosynthesis. SmPALl, SmPAL3, SmC4H1, Sm4CL3, Sm4CL-likel, Sm4CL-like4, SmTATl, SmHPPR3, SmRAS and SmCYP98A78 related to rosmarinic acid biosynthesis exhibited low expression in the periderm and high expression in the phloem and xylem, in accord with the distribution of salvianolic acid. The content of salvianolic acid B in the phloem and xylem was five times higher than that in the periderm. We herein demonstrate that salvianolic acid mainly accumulates and synthesizes in the phloem and xylem of roots. Based on co-expression analysis,2 CYP450s were predicted to catalyze 4-hydroxyohenyllactic acid to 3,4-dihydroxyphenyllactic acid. To explore the reactions that convert rosmarinic acid to salvianolic acids,5 LACs were identified as exhibiting higher expression in the phloem and xylem than in the periderm. Furthermore, our results clearly detected and predicted 26 full-length transcripts and 11 alternative splicing events related to salvianolic acid biosynthesis.4. Identification of poly(A) lncRNA. Here,13,928 lncRNA were identified based on the transcriptomic data from various root tissues in S. miltiorrhiza. Considering the different expression in accordance with distribution of active compounds, we predicted 44 and 51 lncRNAs related to tanshinone and salvianolic acid biosynthesis, respectively.The combination of multidisciplinary technologies, the platform of medicinal model plant, S. miltiorrhiza, would be an efficient pathway to solve the molecular mechanism and regulation networks of active compounds. The synthetic biology of natural products provides the new approach for the challenge of wild resource and artificial cultivation. This medicinal model plant system will also provide reference for selective breeding of fine varieties, and accelerate the TCM development. |
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