| 研究課題/領域番号 |
19K16391
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| 研究機関 | 千葉大学 |
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研究代表者 |
ライ アミット 千葉大学, 大学院薬学研究院, 特任助教 (60760535)
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| 研究期間 (年度) |
2019-04-01 – 2022-03-31
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| キーワード | Metabolic gene cluster / OpCYP1 / MIAs / Pan-Genome / Genome analysis / Metabolite profiling / Specialized metabolites / Camptothecin |
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| 研究実績の概要 |
The key findings are as follows. (1) Pan-genome for MIA producing plants was established using recently assembled plant genome of O. pumila with three other MIA producing plants; (2) The pan-genome analysis identified 33 metabolic gene clusters representing all functionally characterized genes and candidate genes associated with MIA and CPT biosynthesis in plants; (3) Comparative genome analysis showed biosynthesis of strictosidine, which eventually derived MIAs and CPT biosynthesis, evolved through an ancient genome modification. Loss of enzyme catalyzing strictosidine biosynthesis was the key event for the evolution of MIAs and camptothecin biosynthesis; (4) Our analysis also identified several candidate genes that are involved in the biosynthesis of CPT. One of the genes, named as OpCYP1, were selected for functional characterization.; (5) We created a knock-out line for the STR gene, an enzyme that catalyzes the biosynthesis of strictosidine. Knocking out STR resulted in the down-regulation of all MIAs synthesized in O. pumila; (6) Metabolite profiling for STR knock-out lines was compared with identified and putatively assigned MIAs of O. pumila. The comparison showed several metabolite intermediates that could be a member of CPT biosynthesis pathways.; (7) Complete carbon labeling, nitrogen labeling, and tryptophan based metabolome labeling of O. pumila allowed to identify 273 metabolites, assigned with chemical identity based on MS/MS analysis as well as assigning chemical formula based on identified number of carbon atoms, nitrogen atoms, and accurate mass analysis.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We managed to achieve several objectives in the first year of this project. Using comparative genome analysis, we were able to identify several strong candidate genes putatively associated with MIAs biosynthesis. We also used the isotope labeling approach to identify and putatively assign chemical identities to 273 metabolites in Ophiorrhiza pumila. We managed to identify candidate genes and metabolites that we will work in the next two years to confirm their associations in MIA biosynthesis. While we achieve significant progress, we also got slightly delayed. In the proposed research objectives, we mentioned that we would perform comparative genome analysis of monoterpene indole alkaloids producing plants, including O. japonica. While preparing genomic DNA for sequencing Ophiorrhiza japonica, we realized that we would need to acquire more plants to meet the required genomic DNA amount for PacBio based sequencing. Unfortunately, due to the Coronavirus pandemic, it is challenging to source the plant. Initially, we planned to acquire Ophiorrhiza japonica from Okinawa island through our collaborators. However, we will need to wait for a few more months in order to get access to this plant. Similarly, by the end of February 2020, we prepared OpCYP1 knock-out lines for metabolome analysis. However, due to the pandemic, our analysis got delayed. Our first study, comparative genome analysis and genome assembly of O. pumila, is ready as a manuscript, and we are submitting this manuscript to a reputed journal for publication.
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| 今後の研究の推進方策 |
Previous studies have shown activation of specialized metabolites biosynthesis in plants when subjected to phytohormones treatment. We performed phytohormone treatment using Ophiorrhiza pumila hairy root system as a time-series experiment using jasmonic acid, ethephon, abscisic acid, and salicylic acid. We performed the treatment on three weeks old hairy roots, and collected samples for eight-time points, five biological replicates each. We have picked one biological replicate for RNA extraction and transcriptome expression analysis. The rest four biological replicates for each condition will be used for metabolite profiling (targeted and untargeted analysis). This year, we will perform transcriptome analysis, co-expression analysis, metabolome analysis, and integrative omics analysis to identify candidate genes associated with MIA biosynthesis. As our main objective is to understand the biosynthesis and regulation of MIA and camptothecin in O. pumila, this analysis will expand our knowledge. It will help to identify novel genes that could be targeted for functional characterization. We will also look for what fraction of these genes are part of MIA gene clusters, and the positive hits will be chosen for further analysis. We will also work towards establishing a whole-genome assembly for O. japonica, as proposed in our research project. From acquiring plants to the extraction of high molecular weight genomic DNA to whole-genome sequencing and genome assembly will be achieved by this year.
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| 次年度使用額が生じた理由 |
Several of the propose expenditure request were made on the assumption that we will be able to extract high molecular weight genomic DNA for sequencing. Due to unavailability of enough plant sample, we were not able to use the sequencing services proposed in our project last year, and will be performed this financial year. Similarly, request to expand our ability for data storage and cost for consumables will be used this year.
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