| Project/Area Number |
12558075
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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 | 展開研究 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | CHIBA UNIVERSITY |
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Principal Investigator |
SAITO Kazuki Chiba University, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学研究院, 教授 (00146705)
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| Co-Investigator(Kenkyū-buntansha) |
HIGASHI Yousuke Mitsui Chemical, Co., Life Science Research Institute, Researcher, 主任研究員
AIMI Norio Chiba University, Graduate School of Pharmaceutical Sciences, Professor, 大学院・薬学研究院, 教授 (30009170)
YAMAZAKI Mami Chiba University, Graduate School of Pharmaceutical Sciences, Assoc, Progessor, 大学院・薬学研究院, 助教授 (70222370)
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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 |
¥12,300,000 (Direct Cost: ¥12,300,000)
Fiscal Year 2002: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2000: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | Anti-tumor alkaloid / Camptothecin / Ophiorrhiza pumila / hairy root / Agrobacterium rhizogenes / Strictosidine synthase / メタボローム / メタボロミクス / 大量培養 / 細胞内局在 / シロイヌナズナ / RAPD法 / アルカロイド / 抗腫瘍性 |
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| Research Abstract |
Camptothecin derivatives are clinically used anti-tumor alkaloids. Transformed plant cell cultures may be an alternative source of production of camptothecin and related compounds, although they are now producted by extraction from intact plants. We have established a hairy root culture of Ophiorriza pumila (Rubiaceae) incited by Agrobacteriun rhizogenes strain 15834. This hairy root grew well 16-fold times for 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amount of camptothecin. The camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20), to which camptothecin was absorbed. Camptothecin was easily recovered from the resin. Our established method is the most feasible and commercially applicable way to produce camptothecin by in vitro cell culture. We have cloned and characterized cDNAs encoding strictosidine synthase (OpSTR ; EC4.3. 3.2) and tryptophan decarboxylase (OpTDC ; EC4.1.1. 28), two key enzymes in the biosynthesis of camptothecin from hairy roots of 0. Pumila. We also idolated the cDNA coding for NADPH : cytochrome P450 reductase (OpCPR ; EC1. 6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSRC and OpTDC were quite similar, unlike those of OpCPR The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid (SA) repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.
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