| Project/Area Number |
07554045
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
植物生理
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| Research Institution | Nagoya University |
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Principal Investigator |
KONDO Takao Nagoya University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (10124223)
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| Co-Investigator(Kenkyū-buntansha) |
ISHINO Yoshizumi Institute for Biomolecular engineering Research Leader, 中央研究所, 主任研究員
KATO Akira Hokkaido Agriculture Station Research Leader, 作物開発部, 研究室長
ISHIURA Masahiro Nagoya University, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (20132730)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1996: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1995: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | Biological Clock / clock gene / cyanobacteria / circadian rhythm / luciferase / bioluminescence / mutant / environment / 遺伝子発現 / スクリーニング / リアルタイムモニター |
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| Research Abstract |
To expand a potential of luciferase gene as a reporter of gene expression and technology for screening by bioluminescence with a cooled-CCD camera for a various phenomena that living organisms show, we planed this research project. Luciferase reporter library of promoters in a genome of cyanobacteria Synechococcus sp.PCC 7942 contained DNA fragments that were too long for further analysis. Therefore, we re-constructed a genomic library with luciferase reporter gene. Average DNA fragment of new library was 600 bp and the library contained independent E.coil clone as much as to cover the genome of Synechococcus for five times. We screened for 100,000 Synechococcus transformants that were introduced with this DNA-lux constructs and obtained 800 bioluminescent clones. All of 800 clones displayd a circadian bioluminescence rhythms, that confirmed previous results that circadian clock of cyanobacteria controlled most of gene expressions.
Then, we treated the 800 clones with various environmen
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tal stimulus, such as high temperature, low temperature, alteration in light fluence rate, irradiation of monochromatic light including UV range and selected for clones that displayd unique response. Response of clones as monitored with bioluminescence was similar for most of clones but we found several clones of which response was exceptional. We will analyze the response of these selected clones in detail and analyze the promoters that respond to each stimulus.
We sequenced DNA fragments of 10 clones of which bioluminescence was tightly controlled by the circadian clock and search for homology of insert in the genome of Synechocystis (Kazusa DNA Research Institute). Six genes were identified as clock controlled genes and will analyze physiological significance of the control in relation to environmental responses of these genes. Homologous genes will also be search in higher plant to examined clock control and environmental response of these genes in plants.
In addition to luciferase reporter gene, we demonstrated that aequorin genes which was introduced into higher plant can report free Ca level in plants cells and found that Ca level was under control of the circadian clock and light conditions. Less
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