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Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq.

ABSTRACT: Dendrobium officinale L. is an important traditional herb with high commercial value in China. Several bioactive constituents, including polysaccharides and alkaloids, reportedly make major contributions toward the excellent medicinal effect of D. officinale. In this study, the contents of polysaccharides and alkaloids in various organs of D. officinale were measured and compared. We took advantage of transcriptomes from four organs to explore biological mechanisms in the organ-specific distribution of active ingredients in D. officinale. Based on Kyoto Encyclopedia of Genes and Genomes pathways, unigenes related to the enzymes involved in fructose and mannose metabolism and unigenes associated with putative upstream elements of the alkaloid biosynthetic pathway were identified. A large number of candidates, including 35 full-length glycosyltransferase genes and 49 full-length P450 genes, were also identified based on the transcriptome data, and the organ-specific expression pattern of these genes was determined. Furthermore, differential expression of all candidate genes was analyzed in two Dendrobium species, D. nobile L. and D. officinale. The data will supply important clues to exploit useful genes involved in polysaccharide and alkaloid synthesis.

SUBMITTER: Shen C

PROVIDER: S-EPMC5412657 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq.

Shen Chenjia C Guo Hong H Chen Hailing H Shi Yujun Y Meng Yijun Y Lu Jiangjie J Feng Shangguo S Wang Huizhong H

Scientific reports 20170315 1

Dendrobium officinale L. is an important traditional herb with high commercial value in China. Several bioactive constituents, including polysaccharides and alkaloids, reportedly make major contributions toward the excellent medicinal effect of D. officinale. In this study, the contents of polysaccharides and alkaloids in various organs of D. officinale were measured and compared. We took advantage of transcriptomes from four organs to explore biological mechanisms in the organ-specific distribu ...[more]

PMID: 28298629

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Project description:PurposeThere has been mounting evidence that Dendrobium officinale polysaccharides (DOP), a traditional Chinese medicine, are a potential candidate treatment for N-methyl-N'-nitro-N-nitrosoguanidine- (MNNG-) induced precancerous lesions of gastric cancer (PLGC). However, the underlying mechanisms have not been adequately addressed.MethodWe utilized RNA-Seq analysis to investigate possible molecular targets and then used Venn software to identify the differentially expressed genes (DEGs). Further, we analyzed these DEGs with core analysis, upstream analysis, and interaction network analysis by IPA software and validated the DEGs by real-time PCR and Western blot.Result78 DEGs were identified from the normal control group (CON), the PLGC model group (MOD), and the DOP-treated group (DOP) by the Venn software. Further analysis of these DEGs, including core analysis, upstream analysis, and interaction network analysis, was performed by Ingenuity Pathway Analysis (IPA). The main canonical pathways involved were SPINK1 Pancreatic Cancer Pathway (-log (P value) = 4.45, ratio = 0.0667) and Circadian Rhythm Signaling (-log (P value) = 2.33, ratio = 0.0606). Circadian Rhythm Signaling was strongly upregulated in the model group versus the DOP group. CLOCK was predicted to be strongly activated (z-score = 2.236) in upstream analysis and induced the downstream PER3. In addition, the relative mRNA expression levels of seven DEGs (CD2AP, ECM1, AQP4, PER3, CMTM4, ESRRG, and KCNJ15) from RT-PCR agreed with RNA-Seq data from MOD versus CON and MOD versus DOP groups. The gene and protein expression levels of PER3 and AQP4 were significantly downregulated in the PLGC model and significantly increased by DOP treatment (9.6 g/kg).ConclusionsThese findings not only showed DOP inhibits PLGC development by upregulating the PER3 and AQP4 gene and protein expression but also suggested that its mechanism of action involved modulating the Circadian Rhythm Signaling pathway.

Project description:Background: Dendrobium officinale, an endangered Chinese herb, has extensive therapeutic effects and contains bioactive ingredients including a large number of polysaccharides and alkaloids, and minimal flavonoids. Firstly, this study attempts to obtain the protocorm-like bodies of this plant through tissue culture to produce the main secondary metabolites whose distribution in each organelle and protocorm like bodies is analyzed. Then, analysis of the correlation between comparative transcriptome sequence and the metabolite content in different organs enables the discovery of putative genes encoding enzymes involved in the biosynthesis of polysaccharides and alkaloids, and flavonoids. Results: The optimum condition for protocorm-like bodies (PLBs) induction and propagation of D. officinale is established. For protocorm induction, we use the seed as the explant, and the optimum medium formula for PLBs propagation is 1/2 MS + α-NAA 0.5 mg·L-1 +6-BA 1.0 mg·L-1 + 2, 4-D 1.5-2.0 mg·L-1 + potato juice 100 g·L-1. The distribution of polysaccharides, alkaloids and flavonoids in D. officinale organs was clarified. Stems, PLBs and leaves have the highest content of polysaccharides, alkaloids and flavonoids, respectively. PLBs replace organs to produce alkaloids in D. officinale, and naringenin was only produced in stem. Hot water extraction (HWE) method was found outperforming the ultrasound-assisted extraction (UAE) method for polysaccharides from D. officinale. A comparative transcriptome analysis of the protocorm-like bodies and leaves of D. officinale showed genes encoding enzymes involved in polysaccharides, alkaloids and flavonoids biosynthetic pathway were differentially expressed. Putative genes encoding enzymes involved in polysaccharides, alkaloids and flavonoids synthetic pathway were identified. Notably, genes encoding enzymes of strictosidine beta-glucosidase, geissoschizine synthase and vinorine synthase in alkaloids biosynthesis of D. officinale are first reported. Conclusions: Our works, especially the identification of candidate genes encoding enzymes involved in metabolites biosynthesis will help to explore and protect the endangered genetic resources and will also facilitate further analysis of the molecular mechanism of secondary metabolites’ biosynthesis in D. officinale.

Project description:Introduction: Numerous studies have demonstrated that the stems of D. officinale have the effect of lowering blood glucose, but the leaves of D. officinale have seldom been investigated. In this study, we mainly studied the hypoglycemic effect and mechanism of D. officinale leaves. Methods: Initially in vivo, male C57BL/6 mice were administered either standard feed (10 kcal% fat) or high-fat feed (60 kcal% fat) along with either normal drinking water or drinking water containing 5 g/L water extract of D. officinale leaves (EDL) for 16 weeks, and changes in body weight, food intake, blood glucose, etc., were monitored weekly. Next in vitro, C2C12 myofiber precursor cells which were induced to differentiate into myofibroblasts and cultured with EDL to detect the expression of insulin signaling pathway related proteins. HEPA cells were also cultured with EDL to detect the expression of hepatic gluconeogenesis or hepatic glycogen synthesis related proteins. Eventually after separating the components from EDL by ethanol and 3 kDa ultrafiltration centrifuge tube, we conducted animal experiments using the ethanol-soluble fraction of EDL (ESFE), ethanol-insoluble fraction of EDL (EIFE), ESFE with a molecular weight of >3 kDa (>3 kDa ESFE), and ESFE with a molecular weight of <3 kDa (<3 kDa ESFE) for intensive study. Results: The results in vivo revealed that the mice fed the high-fat diet exhibited significantly decreased blood glucose levels and significantly increased glucose tolerance after the EDL treatment, whereas the mice fed the low-fat diet did not. The results in vitro showed that EDL activated the expression of protein kinase B (AKT), the phosphorylation of AKT, and the expression of downstream GSK3β in the insulin signaling pathway. EDL treatment of HEPA cells confirmed that EDL did not affect hepatic gluconeogenesis or hepatic glycogen synthesis. In the experiment of studying the composition of EDL, we found that the >3 kDa ESFE displayed the effect of lowering blood glucose. In summary, the effect of EDL in lowering blood glucose may bethanole achieved by activating the insulin signaling pathway to increase insulin sensitivity, and the main functional substance was contained within the >3 kDa ESFE. Discussion: The findings of this study represent a reference point for further exploration of the hypoglycemic effects of D. officinale leaves and may assist in both the identification of new molecular mechanisms to improve insulin sensitivity and the isolation of monomeric substances that lower blood glucose. Furthermore, the obtained results may provide a theoretical basis for the development of hypoglycemic drugs with D. officinale leaves as the main component.

Project description:BackgroundDendrobium officinale, an endangered Chinese herb, possesses extensive therapeutic effects and contains bioactive ingredients such as major polysaccharides, alkaloids, and minimal flavonoids. We first obtained the protocorm-like bodies (PLBs) of this plant through tissue culture in order to determine the distribution of the main secondary metabolites in each organelle and the PLBs. We then analyzed the correlation between gene expression level from comparative transcriptome sequencing and metabolite content in different organs to identify putative genes encoding enzymes involved in the biosynthesis of polysaccharides, alkaloids, and flavonoids.ResultsWe used seeds as explants for protocorm induction and PLB propagation of D. officinale. The optimal medium formula for PLB propagation was 1/2 MS + α-NAA 0.5 mg·L- 1 + 6-BA 1.0 mg·L- 1 + 2, 4-D 1.5-2.0 mg·L- 1 + potato juice 100 g·L- 1. Stems, PLBs and leaves of D. officinale had the highest content of polysaccharides, alkaloids and flavonoids, respectively. Naringenin was only produced in stem; however, PLBs with high alkaloid content can replace other organs producing alkaloids. The hot water extraction method outperformed the ultrasound-assisted extraction method for extracting polysaccharides from D. officinale. A comparative transcriptome analysis of PLBs and leaves of D. officinale revealed differential expression of genes encoding enzymes involved in polysaccharide, alkaloid and flavonoid biosynthetic pathways. Putative genes encoding enzymes involved in these biosynthetic pathways were identified. Notably, we identified genes encoding the alkaloid biosynthesis enzymes strictosidine β-D-Glucosidase, geissoschizine synthase and vinorine synthase in D. officinale.ConclusionsThe identification of candidate genes encoding enzymes involved in metabolite biosynthesis will help to explore and protect this endangered species and facilitate further analysis of the molecular mechanism of secondary metabolite biosynthesis in D. officinale.

Dataset Information

Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq.

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Identification and analysis of genes associated with the synthesis of bioactive constituents in Dendrobium officinale using RNA-Seq.

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