| Project/Area Number |
12460034
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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 |
Plant nutrition/Soil science
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| Research Institution | Osaka Prefecture University |
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Principal Investigator |
TAKAHASHI Masaaki Osaka Prefecture University, Graduate school of agriculture and life sciences, professor, 農学生命科学研究科, 教授 (30027198)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIURA Miwa Osaka Prefecture University, Graduate school of agriculture and life sciences, assistant professor, 農学生命科学研究科, 助手 (80312255)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥9,600,000 (Direct Cost: ¥9,600,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2000: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Keywords | germin-like protein / transgenic plant / green fluorescent protein (GFP) / GUS staining / target sequence / metal ion channel / site-directed mutagenesis / oxalate oxidase / トマト / 金属イオン輸送 / GFP / germin / 微量必須金属イオン / レステイン / 金属結合 / 遺伝子導入 / germin-like protein / 金属吸収 / 会合 / 配位子 / システイン |
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| Research Abstract |
In our research project following novel findings were obtained on the function of a metal-binding protein, LeGlp 1, in tomato root.
(1)Recombinant LeGlp 1 was purified by metal affinity chromatography and used as antigen to raise antibodies. Antibodies were very specific to LeGlp 1 which enables to quantitatively detect LeGlp 1.
(2)Metal binding of LeGlp 1 was shown for its monomeric and hexameric forms.
(3)In the leaves of LeGlp 1 transgenic tobacco LeGlp 1 was expressed with a weak oxalate oxidase activity in a form that is identical with the native LeGlp 1 in tomato roots.
(4)GUS and GFP were localized in the roots of transgenic tobacco that had been constructed to express either of the reporter proteins fused their N-terminal with the target sequence of LeGlp 1. According to microscopic observation LeGlp 1 may be targeted to plasma membrane of cortex or endoderm cell in roots and may not be secreted to apoplast.
(5)By a site-directed mutagenesis of Cys residues to Ser in the region of newly identified metal binding site at the N-terminal of LeGlp 1, mutated LeGlp 1 was shown to fail in the construction of hexameric natural form of LeGlp 1 in transgenic tobacco leaves.
(6)Protein fraction of LeGlp 1 transgenic tobacco leaves suppressed growth of E.coli. Since this activity of LeGlp 1 was lowered by the substitution of metal-binding Cys residues to Ser of LeGlp 1, LeGlp 1 was suggested to be a metal ion channel at the plasma membrane of E.coli which resulted in the disorder of metal homeostasis in the bacterial cells.
In conclusion, LeGlp 1 is shown to function at the plasma membrane of cortex or endoderm cells as a metal ion channel in the rout of micronutrient uptake from apoplast to xylem tube.
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