- gracile) is an annual herb with pharmacological properties effective in the treatment of various diseases, including hepatic carcinoma. - However, the molecular understanding of the triterpenoid saponin biosynthesis pathway remains unclear.. - Results: In this study, we performed RNA sequencing (RNA-Seq) analysis of the flowers, leaves, roots, and stems of C. - gracile and provide valuable information for the identification of candidate genes involved in the biosynthesis of triterpenoid saponins and other important secondary metabolites.. - These components were condensed in a sequential manner by prenyltrans- ferases, resulting in the formation of prenyl diphos- phates, such as geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP), which are further. - 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - tional enzyme in the terpenoid biosynthesis pathway. - Thus, RNA-Seq is a powerful tool for acquiring gene expression information and is of great significance in the mining of functional genes, analysis of gene expression profiles, and discovery of genetic metabolic networks [20]. - At present, transcriptome se- quencing has been applied to medicinal plants of the Labiatae family, including Scutellaria baicalensis Georgi [21], Ocimum sanctum and Ocimum basilicum [22], Mentha piperita and Mentha arvensis [23], and Clinopo- dium chinense [24]. - This work lays a foundation for further exploration of the molecular mechanism of triterpenoid saponin biosyn- thesis in C. - gracile data showed that the differ- ences among the total saponin content of the four. - RNA-Seq analysis of C. - Of the 128,856 unigenes unigenes) were longer than 1000 bp, and unigenes) were longer than 1500 bp (Additional file 3: Figure S1).. - Of the 128,856 unigenes and 55.67% mapped to GO, KEGG, KOG, NR, NT, Pfam, and SwissProt databases, respectively.. - A total of and 73,919 unigenes were counted in the RNA-Seq data sets of flower, leaf, stem, and root tissues, of which and 9085 unigenes with FPKM ≥10 showed high expression and 21,844 unigenes with FPKM = 1–10 showed medium expression, and and 42,950 unigenes with FPKM ≤1 showed low expression, respectively (Fig. - Identification of candidate genes involved in the triterpenoid biosynthesis pathway. - A total of 1406 uni- genes were enriched in the phenylpropanoid biosyn- thesis pathway (Fig. - 3 Gene expression profiles in the four tissues of C. - (b) Expressed unigenes in the four tissues by a boxplot. - followed by “Carotenoid biosynthesis,” “Ubiquinone and other terpenoid quinone biosynthesis,” “Diterpen- oid biosynthesis,” and “Sesquiterpenoid and triterpen- oid biosynthesis” (Fig. - A total of 523 unigenes were enriched in the “terpenoid backbone biosynthesis” (ko00900) and “sesquiterpenoid and triterpenoid biosynthesis” (ko00909) pathways. - A total of 108 unigenes encoded 6 key enzymes are involved in the MVA pathway. - 74 unigenes encoded 8 key enzymes of the MEP pathway. - and 99 unigenes encoded 5 key en- zymes involved in the conversion of IPP to β-Amyrin (Table 2). - The expression level of unigenes encoding key enzymes involved in the triterpenoid biosynthesis pathways is shown in the heat map. - Most of the unigenes encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK) and HDR showed the highest expression in leaves, while most of the unigenes encoding acetyl-CoA acetyltransferase. - We analyzed the structure of the CL12163–4 unigene, as it showed the highest expression in leaves. - In the multiple sequence align- ment of these SS amino acid sequences, six domains (I–. - The secondary structure of CgSS contained 18 alpha helixes, which were the main component of the SS enzyme (Fig. - The 3D model of CgSS was constructed on the basis of the crystal structure of SS from Trypanosoma cruzi (PDB ID 3wca.3.A), which shares 46.02% sequence iden- tity with CgSS [27]. - Expression levels of unigenes encoding enzymes that catalyze each step of the triterpenoid saponin biosynthesis pathway are shown. - CL ” and “ Un ” indicates a cluster of transcripts and unigenes, respectively. - the expression levels of CL12163–4 (SS), and CL1648–1 (HDR) were the highest in leaves, while that of Un 17, 275 (HMGR) was the highest in flowers. - The 37,744 DEGs identified in the leaves vs. - flowers comparison were mainly enriched in “Indole alkaloid bio- synthesis,” “Photosynthesis,” “Taurine and hypotaurine me- tabolism,” “Riboflavin metabolism,” and “Glycosphingolipid biosynthesis globo and isoglobo series” (Fig. - The 59, 154 DEGs identified in the leaves vs. - roots comparison were primarily enriched in “Photosynthesis,” “Circadian. - rhythm plant,” “Photosynthesis antenna proteins,” “Brassi- nosteroid biosynthesis,” and “Biosynthesis of unsaturated fatty acids” (Fig. - The 36,467 DEGs identified in the leaves vs. - “Photosynthesis,” “Cyanoamino acid metabolism,” and. - 7 Tertiary structure model and schematic diagram of the CgSS active site. - a Cartoon display of the three-dimensional structure of CgSS and five conserved domains (I: red, II: green, III: blue, IV: yellow, V: magenta). - 8 Quantitative real-time PCR (qRT-PCR) analysis of six unigenes encoding enzymes involved in the triterpenoid biosynthesis pathway in C.. - Blue bars show the results of the qRT-PCR analysis, and red lines show the FPKM values determined by RNA-Seq analysis. - The left Y-axis indicates the relative expression level of the qRT-PCR detection gene, and the right Y-axis indicates the FPKM value in the RNA-Seq data. - 1) were mapped to 132 pathways in the KEGG database. - these DEGs were mainly enriched in “Photosynthesis,” “Photosynthesis-antenna proteins,”. - In the GO database, unigenes with rhizome-specific expression were mapped to 49 subcategories in three functional cat- egories, with the highest enrichment in “metabolic process,” “cellular process,” and “biological regulation”. - “membrane,” “membrane part,” and “cell” under “cellular component”. - and “cata- lytic activity,” “binding,” and “transporter” activity under. - A total of 3536 unigenes encoding TFs were identified in the C. - A total of 23 unigenes encoding TF participated in the bio- synthesis of secondary metabolites (Additional file 8:. - Of the 128,856 unigenes, 99,020 were annotated, whereas 23.15% of the unigenes. - b Number of DEGs identified in the leaves compared with roots, flowers, and stems of C. - gracile transcriptome was better than that of other medicinal plants in the Labiatae family such as Red Perilla frutescens var. - of unigenes = 61,400. - of unigenes = 50,778;. - These results demonstrate the reliability of the C. - We performed transcriptome analysis of the flowers, leaves, roots, and stems of C. - gracile to facilitate the dissec- tion of genes involved in the tissue-specific biosynthesis of triterpenoid saponins. - This approach is widely used for the identification of novel genes involved in the biosynthesis of secondary metabolites and analysis of the molecular mech- anism of triterpenoid saponin biosynthesis [31].. - In the transcriptome data sets of C. - “Terpenoid backbone biosynthesis” and “Sesquiterpenoid and triterpenoid biosynthesis” in the KEGG database and were mainly annotated under “metabolic process”. - and “catalytic activity” in the GO database. - These up- regulated genes may explain the molecular basis of the medicinal value of C. - Tri- terpenoid saponins are some of the most important compo- nents of C. - (a – c) Significantly enriched pathways in the DEGs identified in the comparison between leaves vs. - Higher expression levels of some of the unigenes encoding SS, HDR, HDS, DXS, and DXR enzymes in leaves compared with stems, flowers, and roots were consistent with the higher saponin accumulation in C. - A simi- lar approach, i.e., overexpression of the SS gene has been previously used to enhance the production of total sapo- nins in Medicago truncatula [33].. - Roots showed the highest expression of unigenes encod- ing most of the key enzymes involved in the biosynthesis of triterpenoid saponins (Fig. - Multiple sequence alignment of SS amino acid se- quences suggested the presence of six domains (I–VI) required for the functional activity of the enzyme. - Do- mains I, II, and III are involved in the first step of SS biosynthesis. - domain IV participates in the second step;. - 11 KEGG and GO annotations of unigenes expressed specifically in the leaves of C. - In the C. - gracile tran- scriptome, only one unigene (Un 15,683) encoding an MYB TF was annotated to play a role in the metabolism of terpenoids. - Overexpression of the gene encoding MYB1 and MYB2 TFs enhances the synthesis and accumulation of anthocyanins in Fagopyrum tataricum [43]. - These TFs may also play a signifi- cant role in the regulation of triterpenoid saponins and other secondary metabolites in C. - gracile tissues to identify genes in- volved in the biosynthesis of triterpenoid saponins. - The transcriptome data will facilitate further examination of the molecular mechanism of triterpenoid saponin bio- synthesis in C. - 12 The number of unigenes encoding TFs involved in the metabolic pathways in C. - 18 cm) were har- vested from the herbal garden of the Anhui University of Chinese Medicine under the permission of managers and professionals on April 18, 2018 (Additional file 9:. - The absorbance of the solution was de- termined by UV-spectrophotometry (JASCO Company, Japan). - The Xevo G2-S Q-TOF mass spectrometer was run in the negative mode. - De novo assembly of the high-quality reads was performed using Trinity [48]. - Annotation of unigenes. - Conserved domains in the amino acid sequence of CgSS were detected using the Conserved Domains Database (http://www.ncbi.nlm.nih.gov/Struc- ture/cdd/wrpsb.cgi. - Analysis of the key genes expression level in triterpenoid saponin biosynthesis by qRT-PCR. - Identification of the constituents of triterpenoid saponins in C.gracile by UPLC/Q-TOF-MS.. - KEGG functional classification of the annotated unigenes in C. - Number of unigenes encoding TFs involved in terpenoid metabolism.. - gracile in the herbal garden of the Anhui University of Chinese Medicine on April 18, 2018.. - (a) The ultraviolet Absorption Spectrum of the buddlejasaponin IV. - The design of the study was supported by the Sustainable Utilization Project of Chinese Medicine Resources (Grant No. - gracile have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession number SRP194041.. - GC-MS analysis of the volatile oil from Clinopodium Gracile. - cDNA cloning, mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus. - Deep sequencing of the Scutellaria baicalensis Georgi Transcriptome reveals flavonoid biosynthetic profiling and organ-specific gene expression. - Identification and expression analysis under abiotic stress of the R2R3 - MYB genes in Ginkgo biloba L
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