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pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco

Authors

  • Marketa Samalova,

    1. Department of Plant Sciences, University of Oxford, South Parks Rd, Oxford OX1 3RB, UK
    2. Department of Plant Physiology and Anatomy, Faculty of Science, Masaryk University, Kotlarska 2, CZ-61265 Brno, Czech Republic
    3. Institute of Biophysics AS CR, Kralovopolska 135, CZ-61265 Brno, Czech Republic
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  • Bretislav Brzobohaty,

    1. Institute of Biophysics AS CR, Kralovopolska 135, CZ-61265 Brno, Czech Republic
    2. Department of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Kotlarska 2, CZ-61265 Brno, Czech Republic
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  • Ian Moore

    Corresponding author
    1. Department of Plant Sciences, University of Oxford, South Parks Rd, Oxford OX1 3RB, UK
      (fax +44 0 1865 275074; e-mail ian.moore@plants.ox.ac.uk).
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(fax +44 0 1865 275074; e-mail ian.moore@plants.ox.ac.uk).

Summary

We describe pOp/LhGR, a dexamethasone-inducible derivative of the pOp/LhG4 transcription activation system, and its use in tobacco to regulate expression of uidA (encoding β-glucuronidase; GUS) and the cytokinin-biosnythetic gene ipt. The pOp/LhGR system exhibited stringent regulation and strong induced phenotypes in soil and tissue culture. In conjunction with an improved target promoter, pOp6, that carries six copies of an optimized lac operator sequence the pOp6/LhGR system directed induced GUS activities that exceeded those obtained with pOp/LhG4 or the CaMV 35S promoter but without increased uninduced activity. A single dose of dexamethasone was sufficient to direct cytotoxic levels of ipt expression in soil-grown plants although uninduced plants grew normally throughout a complete life cycle. In vitro, induced transcripts were detectable within an hour of dexamethasone application and 1 nm dexamethasone was sufficient for half maximal induction of GUS activity. Various methods of dexamethasone application were successfully applied under tissue culture and greenhouse conditions. We observed no inhibitory effects of dexamethasone or LhGR on plant development even with the highest concentrations of inducer, although tobacco seedlings were adversely affected by ethanol used as a solvent for dexamethasone stock solutions. The pOp/LhGR system provides a highly sensitive, efficient, and tightly regulated chemically inducible transgene expression system for tobacco plants.

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