|タイトル||*平成29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|
|報告書年度||2017 - 2017|
|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) FY2017 Annual Report
1. Development of Product-accumulating Technology through the Regulation of Gladular Trichome Differentiation
1-1. Promotion of Glandular Trichome Differentiation by Genome Editing
The content of this part is to try the promotion of glandular trichome differentiation in tomato by targeting the genes, which act as negative regulators of trichome differentiation, through genome editing technology.
In the H29 fiscal year, two tomato TRY/CPC homologous genes were broken in transgenic Micro-Tom calli by the CRISPR/Cas9 system. Four CRISPR/Cas9 constructs for each gene were made, and several regenerating plants for each gene were obtained. As the results, in several independent lines the coding sequence of a targeted gene was heterozygously broken for each TRY/CPC homologous gene. The phenotypic analysis is done using single and double mutant plants in their following generations.
1-2. Screening for Chemicals Promoting Gladular Trichome Differentiation
The content of this part is to retrieve chemicals that can promote trichome differentiation from chemical libraries, taking the Arabidopsis reporter line GFP-GL2.
In the H29 fiscal year, a novel method for growing seedlings in the chemical-screening system was developed for a high-throughput screening. Using this system, a drug-derived chemical library that contains ca. 1,000 chemicals with medicinal effects, was screened. As the result, several chemicals including tolnaftate were found to change the fluorescence signal strength of GFP-GL2 in roots. The large-scale screening is continued.
2. Development of Product-accumulating Technology through the Regulation of Transport Machinery
2-1. Molecular Dissection of Intracellular Transport Cargo for Lipophilic Metabolites
The production of highly hydrophobic shikonin derivatives by Lithospermum erythrorhizon cells is inducible by the M9 medium and these lipid-soluble compounds are exclusively secreted out of the cells, while the production is strongly inhibited by light irradiation.
By de novo RNA-Seq and a comprehensive proteome analysis with nano LC-MS/MS, a series of shikonin producing cell-specific genes/proteins were found. In the H29 fiscal year, we selected fourteen strong candidates, which may be involved in the secretion process or formation of ‘transport cargo’ of lipophilic metabolites in this plant. These candidates were subcloned in an entry vector for future studies.
As a maker of shikonin transporting cargo, a small polypeptide LeDI2 was selected and its subcellular localization was shown to be endoplasmic reticulum, at least in part. The lipid molecules co-existing with shikonin derivatives were also identified by LC-MS and GC-FID.
2-2. Application of Key Components involved in Transport Complex
This program will be performed after 2019.