A multiple shoot induction system for peptide-mediated gene delivery into plastids in Arabidopsis thaliana, Nicotiana benthamiana, and Fragaria×ananassa

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  • Odahara Masaki
    Biomacromolecule Research Team, RIKEN Center for Sustainable Resource Science
  • Ara Most Tanziman
    Biomacromolecule Research Team, RIKEN Center for Sustainable Resource Science
  • Nakagawa Remi
    Resources Group, Tsukuba Research Institute, Sumitomo Forestry Co., Ltd.
  • Horii Yoko
    Biomacromolecule Research Team, RIKEN Center for Sustainable Resource Science
  • Ishio Shougo
    Resources Group, Tsukuba Research Institute, Sumitomo Forestry Co., Ltd.
  • Ogita Shinjiro
    Department of Local Resources, Faculty of Bioresource Sciences, Prefectural University of Hiroshima
  • Numata Keiji
    Biomacromolecule Research Team, RIKEN Center for Sustainable Resource Science Department of Material Chemistry, Kyoto University

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  • A multiple shoot induction system for peptide-mediated gene delivery into plastids in <i>Arabidopsis thaliana</i>, <i>Nicotiana benthamiana</i>, and <i>Fragaria</i>×<i>ananassa</i>

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<p>The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.</p>

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