| Project/Area Number |
14350436
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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 |
生物・生体工学
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| Research Institution | Nara Institute of Science and Technology |
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Principal Investigator |
YOSHIDA Kazuya Nara Institute of Science and Technology, Graduate School of Biological Sciences, Associate Professor, バイオサイエンス研究科, 助教授 (50252622)
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| Co-Investigator(Kenkyū-buntansha) |
SHINMYO Atsuhiko Nara Institute of Science and Technology, Graduate School of Biological Sciences, Professor, バイオサイエンス研究科, 教授 (30029235)
NAKAYAMA Hideki Nara Institute of Science and Technology, Graduate School of Biological Sciences, Assistant Professor, バイオサイエンス研究科, 助手 (30324982)
SEKI Tatsuji Osaka University, International Center of Biotechnology, Professor, 生物工学国際交流センター, 教授 (50029245)
FUJIYAMA Kazuhito Osaka University, International Center of Biotechnology, Associate Professor, 生物工学国際交流センター, 助教授 (70209112)
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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 |
¥14,100,000 (Direct Cost: ¥14,100,000)
Fiscal Year 2004: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2003: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 2002: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | transgenic plants / vesicular transport / peroxidase / propeptide / glycan structure / horseradish / tobacco / タバコ培養細胞 / EGFP / 小胞体 / 液胞 / タンパク質分泌生産 |
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| Research Abstract |
Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals, and in plants have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein was shown to be synthesized as a preprotein wish both an N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, with using analytic system of transgenic cultured tobacco cells (Nicotiana tabacum, BY2).
In this research, we found out that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. HRP C1a could be secreted from cells expressing HRP C1a gene without the DNA region encoding the CTPP. We also investigated the roles of the NTPP and the CTPP of HRP C1a in the tobacco plant. In transgenic tobacco plants expressing the HRP C1a gene with NTPP and CTPP,HRP C1a was accumulated in the vacuole. When HRP C1a lacking the CTPP was expressed, active enzyme was secreted into the apoplast space. And, we could enhance the expression of HRP C1a by the use of translational enhancer, NtADH 5'-UTR. Furthermore, we determined the glycan structure of recombinant C1a protein in BY2 cells was Man3FucXylGlcNAc which was same structure as that of endogenous vacuolar glycol-proteins.
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