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Basic studies on the chloroplast transformation using photoautotrophic cultured cells

Research Project

Project/Area Number 63560082
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 応用生物化学・栄養化学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

SATO Fumihiko  Research Center for Cell & Tissue. Culture, Instructor, 農学部, 助手 (10127087)

Co-Investigator(Kenkyū-buntansha) YAMADA Yasuyuki  Research Center for Cell & Tissue. Culture, Professor, 農学部, 教授 (50026415)
Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsPhotoautotrophic cultured cells / Chloroplast transformation / Herbicide (atrazine) resistance / recombinant DNA / Electroporation / chloroplast genome / D-1 protein / Chloroplast mutant / Dー1タンパク質 / 光独立栄養細胞 / 除草剤抵抗性 / Direct gene transfer / T_1プラスミドベクター
Research Abstract

Chloroplasts have an important role in photosynthesis, nitrogen metabolism and amino acid biosynthesis and many chloroplast proteins are encoded by the chloroplast genome. However, these properties make the isolation of chloroplast mutants of higher plants difficult ; the mutations are often lethal and the high copy number of the chloroplast genome makes the selection of mutants inefficient. Recently, we have shown that a photomixotrophic cell line of tobacco (Nicotiand tabacum cv Samsun. NN) was very useful to select a cell line resistant to the herbicide atrazine, as a model system for the isolation of chloroplast mutants. Based on this success, the basic studies were carried out to establish the method to transform chloroplast genome.
1. A vector, in which kanamycin resistant gene (NPTII) was cloned downstream the promoter of psbA gene, was constructed to transform chloroplast genome. 2. Another vector, in which psbA gene was placed downstream the promoter and transit peptide of wheat Rubisco small subunit, was constructed to convert chloroplast psbA gene into a nuclear gene. 3. The conditions for the isolation and culture of protoplasts from photomixotrophic cells were established for the direct gene transfer of photomixotrophic cells. Further experiments to transform photomixotrophic cells is in progress. 4. Atrazine-resistant protoplasts and mesophyll protoplasts were fused asymmetrically to regenerate plants with mutant chloroplasts. By this method, putable atrazine-resistant shoots were obtained. 5. To understand the resistance mechanism of mutant cells and the function of D-1 protein, the projected secondary structures of the mutant D1 proteins were analyzed. This result indicates that the cross-resistantce of threonine264 D1 protein to triazine and urea herbicides is mainly due to a conformational change of the binding site for the herbicides.

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] Y.Shigematsu: "The mecanism of herbicide resistance in tobacco cells with a new mutation in the Q_B protein" Plant Physiology. 89. 986-992 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Y.Shigematsu: "A binding model for phenyl urea-herbicides based on analysis of a Thr264 mutation in the D-1 protein of tobacco" Pesticide Biochemistry and Physiology. 35. 33-41 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] F.Sato: "Photoagtotrophic culture:A Model system to investigate the regulation of gene expression in cultured plant cells" In“Primary and secondary metabolism of plant cell culture II",ed.by W.G.W.Kurz,Springer-Verlag. 27-34 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 佐藤文彦: "植物培養細胞における炭酸固定機能に関する研究" 日本農芸化学会誌. 63. 1855-1861 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Y. Shigematsu et al.: "The mechanism of herbicide resistance in tobacco cells with a new mutation in the Q_B protein" Plant Physiol., 89: 986-992 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Y. Shigematsu et al.: "A binding model for phenyl ureaherbicides based on analysis of a Thr264 mutation in the D-1 protein of Tobacco" Pesticide Biochemistry & Physiology, 35: 33-41 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] F. Sato et al.: "Photoautotrophic culture: A model system to investigate the regulation of gene expression in cultured plant cells" In "Primary and secondary metabolism of plant cell cultureII ed. by G.W.G. Kurz, Springer-Verla Berlin, pp27-34, 1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] F. Sato: "Photoautotrophism and its application in cultured plant cells (in japanese)" Nippon Nogeikagaku Kaishi 63: 1855-1861 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Y.Shigematsu: "The mechanism of herbicide resistance in tobacco cells with a new mutation in the O_B protein" Plant Physiology. 89. 986-992 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Y.Shigematsu: "A binding model for phehyl ureaーherbicides based on analysis of a Thr264 mutation in the Dー1 protein of tobacco" Pesticide Biochemistry and Physiology. 35. 33-41 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] F.Sato: "Photoautotrophic culture:A model system to investigate the regulation of gene expression in cultured plant cells" In“Primary and secondary metabolism of plant cell culture II",ed.by W.G.W.Kurz,SpringerーVerlag,Berlin. 27-34 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 佐藤文彦: "植物培養細胞における炭酸固定機能に関する研究" 日本農芸化学会誌. 63. 1855-1861 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] F.Sato: Mol.Gen.Genet.214. 358-360 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] 佐藤文彦: 組織培養. 15(1). 7-11 (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Y.Shigematsu: "Proceedings of II International Symposium on Primary and Secondary Metabolism of Plant Cell Cultures" Springer-verlag, (1989)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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