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成果報告書詳細
管理番号20170000000565
タイトル*平成28年度中間年報 植物等の生物を用いた高機能品生産技術の開発 植物の生産性制御に係る共通基盤技術開発 植物における代謝産物の蓄積機構の制御技術の開発
公開日2017/7/6
報告書年度2016 - 2016
委託先名国立大学法人京都大学
プロジェクト番号P16009
部署名材料・ナノテクノロジー部
和文要約
英文要約Title: Development of Biotechnology to Produce Industrial Materials with Plants and Microorganisms. Development of Product-accumulating Technology in Plants (FY2016-FY2018) FY2016 Annual Report

1-1. Promotion of Glandular Trichome Differentiation by Genome Editing.The content of implementation in this part is to try the promotion of glandular trichome differentiation in tomato (Solanum lycopersicum) by targeting the genes homologous to the Arabidopsis thaliana R3-MYB type transcription factor genes CPC, TRY, and ETC3, which act as negative regulators of trichome differentiation, taking advantage of the genome editing technology with the CRISPR/Cas9 system.
    In the H28 fiscal year, the tomato genes Solyco1g095640.1 and Solyco7g054290.2 were identified as homologs of Arabidopsis CPC, TRY, and ETC3 by an in silico analysis with the tomato genome sequence database and an RNA-Seq analysis with RNAs prepared from shoot primordia and roots of the tomato dwarf variety Micro-Tom. Then, to obtain tomato mutant lines without the function of Solyco1g095640.1 or Solyco7g054290.2, four CRISPR/Cas9 DNA constructs, which varied in target sequences and Cas9 promoters, were made for each gene. Transgenic Micro-Tom calli were so far obtained with half of the constructs, and the plant regeneration from them are now being performed.
1-2. Screening for Chemicals Promoting Gladular Trichome Differentiation
.The content of implementation in this part is to retrieve chemicals that can promote trichome differentiation from chemical libraries, taking advantage of the Arabidopsis reporter line that expresses a strong fluorescence signal of GFP-GL2 in trichomes.
In the H28 fiscal year, conditions of the chemical library screening were examined experimentally and optimized. In a test run using the optimized conditions, seedlings of the reporter line were grown for one to seven days in wells of a 96-well microplate which contained 20 different compounds at four different concentrations, and then examined for their leaf surface morphology and fluorescence signal of GFP-GL2. Through the test run, the system was established for the chemical library screening starting in the next fiscal year. In addition, the reporter line seeds were propagated for the large-scale screening.
2-1. Molecular Dissection of Intracellular Transport Cargo for Lipophilic Metabolites
.We employ the shikonin producing system of cultured Lithospermum erythrorhizon cells as a model of lipophilic metabolite production by plant cells, which was formerly applied to industrial production of plant secondary metabolites for the first time as a most successful case among plant cell culture systems. Shikonin derivatives are highly hydrophobic metabolites, whose production is inducible by using the pigment production medium M9 where these lipophilic compounds are exclusively secreted to cell surface, while the production is strongly inhibited by light irradiation. Taking these features, a large scale de novo RNA-Seq was done and genes preferentially expressed under shikonin producing conditions were collected. In parallel, we carried out a comprehensive proteome analysis with nano LC-MS/MS, by which red cell-specific protein fragments were picked up. By analyzing the predicted functions of those gene products, both from transcriptomic and proteomic analyses, we selected thirteen strong candidates that may be involved in the secretion process or formation of transport cargo of lipophilic metabolites in L. erythrorhizon; i.e., four from the transcriptome and nine from proteome data.
To apply them for further studies on subcellular localizations and protein-protein interactions, we carried out 5’- and 3’-RACE to clone the full-length cDNAs of those candidates, and after confirmation by sequencing they were successively transferred into entry clones of Gateway system.
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