本文へジャンプ

成果報告書詳細
管理番号20170000000567
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 遺伝子発現制御および栽培環境制御の融合による代謝化合物高生産基盤技術開発 その2
公開日2017/7/6
報告書年度2016 - 2016
委託先名国立大学法人横浜国立大学
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Development of regulated gene expression technologies by transcription factors and expression regulatory elements (FY2016-FY2018) FY2016 Annual Report

We have been working on bioluminescence reporter gene technologies for monitoring regulated gene expression in plants. Based on the technologies, we conducted studies of IRES (internal ribosome entry site) application, intron manipulation, identification of bioactive compounds by chemical screening and the development of artificial transcription factors.
To achieve the requirement, the following sub-projects were conducted in this fiscal year:
1) Improvement and application of multi-gene expression technologies by the use of IRES-mediated gene expression technique.
2) Screening and identification of bioactive chemicals that contribute high-level expression and accumulation of secondary metabolites.
3) Regulated expression of targeted genes by artificial transcription factors in plant cells.
1-1 Improvement of multi-gene expression system by novel intervening sequences
An IRES sequence was used to express RNA silencing suppressors (RSS) in transgenic tobacco seedlings. Transient expression analysis with Agro-infiltration and the bioluminescence reporter assay showed an additive effect of RSS expression mediated by the IRES sequence. This result shows that the transgenic plants can be applicable for the development of platform plants for the production of maximal levels of transgene products.
To improve luciferase bioluminescence reporter assay system, we have been trying to establish a novel multi-reporter system based on the use of luciferase specific inhibitors. In this study, we conducted screening of chemicals that can efficiently inhibit particular luciferase activity at low concentrations. With this technology we demonstrated that three bioluminescence reporters are separable by their sensitivity to inhibitors and their activities can be individually monitor through chemical treatment.
1-2 Screening and the use of agents that enhance high-level expression of genes and the products
High-throughput screening using bioluminescence reporter system was conducted and several low-molecular-weight compounds were identified as candidate of inducer for high-level expression of genes involved in secondary metabolites. Screening of jasmonate-responsive gene inducers from chemical libraries resulted in the identification of novel compounds that may act as methyl jasmonate agonists. Several other stimulants for potent defense-gene inducers including salicylate-responsive gene inducers were also identified.
To improve the screening system, we tested a novel luciferase fusion reporter gene system for monitoring defense gene expression signal transduction pathway. The JAZ1-luciferase protein fusion reporter system was introduced into Arabidopsis and the transgenic seedlings were used to monitor the jasmonate signal transduction pathway.
2-1 Development of artificial transcription factors for regulated expression of genes in plant cells
We invested the use of dCas9-fusion protein system for the assessment of the transcriptional regulatory function of certain transcription factors. The GAL4-UAS system was tested as target for the bioluminescence reporter assay and found that the system can be used for the evaluation of transactivation capability for fusion proteins. Co-expression of guide RNAs complementary to the GAL4-UAS sequence has been shown to be functional in this assay system.
ダウンロード成果報告書データベース(ユーザ登録必須)から、ダウンロードしてください。

▲トップに戻る