Advertisement

Plant Molecular Biology

, Volume 37, Issue 6, pp 1079–1084 | Cite as

Construction of a new vector conferring methotrexate resistance in Nicotiana tabacum plants

  • Tiziana Irdani
  • Patrizia Bogani
  • Alessio Mengoni
  • Giorgio Mastromei
  • Marcello Buiatti
Article

Abstract

A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.

gene expression Candida albicans dihydrofolate reductase dominant selectable marker Nicotiana tabacum 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Baccanari DP, Stone D, Kuyper L: Effect of a single amino acid substitution on Escherichia coli dihydrofolate reductase catalysis and ligand binding. J Biol Chem 256: 1738–1747 (1981).PubMedGoogle Scholar
  2. 2.
    Baccanari DP, Tansik RL, Joyner SS, Fling ME, Smith PL, Freisheim JH: Characterization of Candida albicans dihydrofolate reductase. J Biol Chem 264: 1100–1107 (1989).PubMedGoogle Scholar
  3. 3.
    Becker D, Kemper E, Schell J, Masterson R: New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20: 1195–1197 (1992).PubMedGoogle Scholar
  4. 4.
    Bogani P, Simoni A, Bettini P, Mugnai M, Pellegrini MG, Buiatti M: Genome flux in tomato auto and auxotrophic cell cones cultured in different auxin/cytokinin equilibria. I. DNA multiplicity and metylation levels. Genome 38: 902–912 (1995).Google Scholar
  5. 5.
    Bullock WO, Fernandez JM, Short JM: A high efficiency plasmid transforming recA Escherichia coli strain with betagalactosidase selection. BioTechniques 5: 376 (1987).Google Scholar
  6. 6.
    Daly S, Mastromei G, Yacoub A, Lorenzetti R: Sequence of a dihydrofolate reductase-encoding gene from Candida albicans. Gene 147: 115–118 (1994).PubMedGoogle Scholar
  7. 7.
    Doyle J, Doyle J.: Isolation of plant DNA from fresh tissue. Focus 1: 13–15 (1989).Google Scholar
  8. 8.
    Eichholtz DA, Rogers SG, Horsch RB, Klee HJ, Hayford M, Hoffmann NL, Bradford SB, Fink C, Flick J, O'Connell KM, Fraley RT: Expression of mouse dihydrofolate reductase gene confers methotrexate resistance in transgenic petunia plants. Somatic Cell Mol Genet 13: 67–76 (1987).PubMedGoogle Scholar
  9. 9.
    Franceschi M, Denaro M, Irdani T, Lorenzetti R, Mastromei G, Skarmoutsou E, Polsinelli M: A dihydrofolate reductase gene from Candida albicans molecular cloning. FEMS Microbiol Lett 80: 179–182 (1991).Google Scholar
  10. 10.
    Freisheim JH, Matthews DA: In: Sirotnak FM, Burchall JJ, Ensminger WB, Montgomery JA (eds) Folate antagonists as Therapeutic Agents, vol. 1, pp. 69–131. Academic Press. New York (1983).Google Scholar
  11. 11.
    Guarente L, Mason T: Heme regulates transcription of the CYC1 gene of S. cerevisiae via an upstream activation site. Cell 32: 1279 (1983).PubMedGoogle Scholar
  12. 12.
    Guerineau F, Brooks L, Meadows J, Lucy A, Robinson C, Mullineaux P: Sulfonamide resistance gene for plant transformation. Plant Mol Biol 15: 127–136 (1990).PubMedGoogle Scholar
  13. 13.
    Habert DA, Beverley SM, Kiely ML, Schimke RT: Properties of an altered dihydrofolate reductase encoded by amplified genes in cultures mouse fibroblasts. J Biol Chem 256: 9501–9510 (1981).PubMedGoogle Scholar
  14. 14.
    Herrera-Estrella L, De Block M, Messens E, Hernalsteens JP, Van Montagu M, Schell J: Chimeric genes as dominant selectable markers in plant cells. EMBO J 2: 987–995 (1983).Google Scholar
  15. 15.
    Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary vector strategy based on separation of the Vir-and Tregion of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180 (1980).Google Scholar
  16. 16.
    Horsch RB, Fry JE, Hoffmann NL, Walbroth N, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1129–1331 (1985).Google Scholar
  17. 17.
    Jantzen HM, Strahle U, Gloss B, Stewart F, Schmid W, Boshart M, Miksicek R, Schutz G: Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene. Cell 49: 29–38 (1987).PubMedGoogle Scholar
  18. 18.
    Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).PubMedGoogle Scholar
  19. 19.
    Linsmaier AM, Skoog F: Organic growth factor requirements of tobacco tissue culture. Physiol Plant 18: 100–127 (1965).Google Scholar
  20. 20.
    Little JG, Haynes RH: Isolation and characterization of yeast mutants auxotrophic for 20-deoxythimidine 5′-monophosphate. Mol Gen Genet 168: 141–151 (1979).PubMedGoogle Scholar
  21. 21.
    Luzzati M: Isolation and properties of a thimidylate-less mutant in Saccharomyces cerevisiae. Eur J Biochem 79: 533–538 (1975).Google Scholar
  22. 22.
    Masterson RV, Furtek DB, Grevelding C, Schell J: A maize Ds transposable element containing a dihydrofolate reductase gene transposes in Nicotiana tabacum and Arabidopsis thaliana. Mol Gen Genet 219: 461–466 (1989).CrossRefGoogle Scholar
  23. 23.
    Meijer EGM, Schilperoort RA, Rueb S, van Os-Ruygrok PE, Hensgens LAM: Transgenic rice cell lines and plants: expression of transferred chimeric genes. Plant Mol Biol 16: 807–820 (1991).CrossRefPubMedGoogle Scholar
  24. 24.
    Perez P, Tiraby G, Kallerhoff J, Perret J: Phleomycin resistance as a dominant selectable marker for plant cell transformation. Plant Mol Biol 13: 365–373 (1989).PubMedGoogle Scholar
  25. 25.
    Ratnam S, Delcamp TJ, Hynes JB, Freisheim JH: Purification and characterization of dihydrofolate reductase from soybean seedlings. Arch Biochem Biophys 255: 279–289 (1987).PubMedGoogle Scholar
  26. 26.
    Rogers SG, Klee H, Horsch RB, Fraley RT: Use of cointegrating Ti plasmid vectors. In: Gelvin SB, Schilperoort RA, Verma DPS (eds) Plant Molecular Biology Manual, pp. A2: 1–12. Kluwer Academic Publishers, Dordrecht, Netherlands (1988).Google Scholar
  27. 27.
    Rose MD, Winston F, Hieter P: Methods in Yeast Genetics: Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1991).Google Scholar
  28. 28.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  29. 29.
    Schena M, Yamamoto KR: Mammalian glucocorticoid receptor derivatives enhance transcription in yeast. Science 241: 965–967 (1988).PubMedGoogle Scholar
  30. 30.
    Zeilkson R, Luzzati M: Mitochondrial and cytoplasmic distribution in Saccharomyces cerevisiae of enzymes involved in folate-coenzyme-mediated one carbon-group transfer. A genetic and biochemical study of the enzyme deficiencies in mutants tmp3 and ade3. Eur J Biochem 79: 285–292 (1977).PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Tiziana Irdani
    • 1
  • Patrizia Bogani
    • 2
  • Alessio Mengoni
    • 2
  • Giorgio Mastromei
    • 2
  • Marcello Buiatti
    • 2
  1. 1.Istituto Sperimentale per la Zoologia Agraria, Ministero delle Politiche Agricole, Cascine del RiccioFlorenceItaly
  2. 2.Department of Animal Biology and Genetics ‘Leo Pardi’University of FlorenceFlorenceItaly

Personalised recommendations