| Project/Area Number |
14390030
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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 | 一般 |
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| Research Field |
広領域
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| Research Institution | Kyoto University |
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Principal Investigator |
IZUI Katsura Kyoto University, Graduate School of Biostudy, Professor, 生命科学研究科, 教授 (20025414)
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| Co-Investigator(Kenkyū-buntansha) |
FURUMOTO Tsuyoshi Kyoto University, Graduate School of Biostudy, Assistant Professor, 生命科学研究科, 助手 (30313208)
HISABORI Toru Tokyo Institute of Technology, Chemical Resources Laboratory, Associate Professor, 資源化学研究所, 助教授 (40181094)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥13,100,000 (Direct Cost: ¥13,100,000)
Fiscal Year 2004: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2003: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2002: ¥7,900,000 (Direct Cost: ¥7,900,000)
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| Keywords | phosphoenolpyruvate carboxylas (PEPC). / PEPC kinase / C4 photosynthesis / regulation of enzyme activity by phosphorylation. / recombinant enzyme / ubiquitin / proteasome system / plant transformation / Flaveria / レドックス調節 / ジスルフィド結合 / 一過的発現系 / ユビキチン化 / タンパク質分解調節 / ホスホエノールピルビン酸カルボキシラーゼ / PEPC / プロテインキナーゼ / フラベリア / トウモロコシ / ホスホエノールピルビン酸カルボキチシラーゼ |
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| Research Abstract |
Phosphoenolpyruvate carboxylase (PEPC) which catalyses an initial C0_2 fixation reaction in the C4 photosynthetic pathway, is regulated not only by allosteric effect but also by reversible phosphorylation. For this phosphorylation, a protein kinase whose substrate is confined to PEPC is involved. Thus this protein kinase is called PEPC kinase (PEPCk). We firstly had succeeded in the cloning of PEPCk involved in the C4 photosynthesis from a model C4 plant, Flaveria trinervia, and in its prokaryotic expression. By the use of this PEPCk (FtPEPCk) cDNA, we investigated the regulatory mechanisms of this kinase and evaluated its physiological significance. The results obtained are as described below.
1)At least three copies of the PEPCk genes were found on the genome of Flaveria trinervia, and one of the genes was assigned to be involved in the C4 photosynthesis. The gene was expressed not only in leaves but also in other tissues at low levels.
2)The recombinant FtPEPCk could successfully be obtained as a fusion protein with Nus tag protein. Similar to the case with PEPCk from maize (ZmPEPCk), FtPEPCk was also found to be subject to redox regulation. The candidate Cys residues participating in the disulfide bond formation upon oxidation were assigned.
3)Several amino acid residues of PEPC required for the activation by phosphorylation were identified.
4)By the use of protoplasts of mesophyll cells of maize, FtPEPCk with FLAG tag was transiently expressed and its degradation process was monitored. The Involvement of the ubiquitin/proteasome system in the degradation of FtPEPCk was demonstrated.
5)To evaluate the physiological importance of PEPCk in vivo, PEPCk expression was suppressed by the use of an antisense method with Flaveria bidentis which is a transformable C4 plant. The transformants whose PEPC is not significantly phosphorylated were obtained, and its photosynthetic activities are under investigation.
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