Project description:We analyzed the transcriptome of A. acutangulus roots by deep RNA sequencing to dig TAs biosynthetic genes. KOG (Eukaryotic Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses identified 48 unigenes referring to the tropane, piperidine and pyridine alkaloid biosynthesis, 145 unigenes presumably involved in distribution of arginine to TAs biosynthesis, and 86 unigenes referring to the terpenoid backbone biosynthesis. Furthermore, 82 unigenes annotated as cytochrome P450 family members seemed to be involved in secondary metabolism pathways. Previously unknown TAs biosynthetic genes in A. acutangulus, which encode littorine mutase/monooxygenase (CYP80F1) and diamine oxidase (DAO), were identified by this study.
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:Daphniphyllum macropodum produces alkaloids that are structurally complex with polycyclic, stereochemically rich carbon skeletons. Understanding these compounds are formed by the plant may enable exploration of their biological function and bioactivities.We employed multiple metabolomics techniques, including a workflow to annotate compounds in the absence of standards, to compare alkaloid content across plants and tissues. Different alkaloid structural types were found to have distinct distributions between genotypes, between tissues and within tissues. Alkaloid structural types also showed different isotope labelling enrichments that matched their biosynthetic relationships. The work suggests that mevalonate derived 30-carbon alkaloids are formed in the phloem region prior to their conversion to 22-carbon alkaloids which accumulate in the epidermis, and sets the stage for further investigation into the biosynthetic pathway.
Project description:In previous work, cephalotaxine, harringtonine, homoharringtonine were shown to be accumulated differentially after various stimuli. Especially, after MeJA treatment, the concentration of 3 cephalotaxus alkaloids all showed decreasing. We speculated that the genes expressed lower after MeJA treatment might encode some enzymes responsible for the biosynthesis of cephalotaxus alkaloids. Therefore, choosing the sample treated with MeJA and the control sample for comparative iTRAQ analysis will greatly facilitate dissection of the genes involved in the biosynthesis of cephalotaxus alkaloids and even the acyl portions of cephalotaxus ester alkaloids. This approach is widely used for mining and identifying novel genes in the biosynthesis of secondary metabolites without genome data in plants.