Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

A conditional marker gene allowing both positive and negative selection in plants

Nature Biotechnology volume 22, pages 455–458 (2004)Cite this article

Abstract

Selectable markers enable transgenic plants or cells to be identified after transformation. They can be divided into positive and negative markers conferring a selective advantage or disadvantage, respectively. We present a marker gene, dao1, encoding D-amino acid oxidase (DAAO, EC 1.4.3.3) that can be used for either positive or negative selection, depending on the substrate. DAAO catalyzes the oxidative deamination of a range of D-amino acids1. Selection is based on differences in the toxicity of different D-amino acids and their metabolites to plants. Thus, D-alanine and D-serine are toxic to plants, but are metabolized by DAAO into nontoxic products, whereas D-isoleucine and D-valine have low toxicity, but are metabolized by DAAO into the toxic keto acids 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively. Hence, both positive and negative selection is possible with the same marker gene. The marker has been successfully established in Arabidopsis thaliana, and proven to be versatile, rapidly yielding unambiguous results, and allowing selection immediately after germination.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: D-amino acid dose responses of dao1 transgenic and wild-type A. thaliana.
Figure 2: Selection of primary transformants with the DAAO marker.

Similar content being viewed by others

References

  1. Alonso, J. et al. D-amino acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiol. 144, 1095–1101 (1998).

    Article  CAS  Google Scholar 

  2. Pilone, M.S. D-amino acid oxidase: new findings. Cell. Mol. Life. Sci. 57, 1732–1747 (2000).

    Article  CAS  Google Scholar 

  3. Frommer, W.B., Hummel, S., Unseld, M. & Ninnemann, O. Seed and vascular expression of a high-affinity transporter for cationic amino acids in Arabidopsis. Proc. Natl. Acad. Sci. USA 92, 12036–12040 (1995).

    Article  CAS  Google Scholar 

  4. Boorer, K.J. et al. Kinetics and specificity of a H+/amino acid transporter from Arabidopsis thaliana. J. Biol. Chem. 271, 2213–2220 (1996).

    Article  CAS  Google Scholar 

  5. Umhau, S. et al. The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation. Proc. Natl. Acad. Sci. USA 97, 12463–12468 (2000).

    Article  CAS  Google Scholar 

  6. Yurimoto, H., Hasegawa, T., Sakai, Y. & Kato, N. Physiological role of the D-amino acid oxidase gene DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidini. Yeast 16, 1217–1227 (2000).

    Article  CAS  Google Scholar 

  7. Gabler, M., Hensel, M. & Fischer, L. Detection and substrate selectivity of new microbial D-amino acid oxidases. Enzyme Microb. Techno. 27, 605–611 (2000).

    Article  CAS  Google Scholar 

  8. Lindsey, K. & Gallois, P. Transformation of sugar beet (Beta vulgaris) by Agrobacterium tumefaciens. J. Exp. Bot. 41, 529–536 (1990).

    Article  CAS  Google Scholar 

  9. Gamburg, K.Z. & Rekoslavskaya, N.I. Formation and function of D-amino acids in plants. Soviet Plant Physiol. 38, 904–912 (1991).

    Google Scholar 

  10. Cosloy, S.D. & McFall, E. Metabolism of D-serine in Escherichia coli K-12: Mechanism of growth inhibition. J. Bacteriol. 114, 685–694 (1973).

    Article  CAS  Google Scholar 

  11. Soutourina, J., Plateau, P., Delort, F., Peirotes, A. & Blanquet, S. Functional characterization of the D-Tyr-tRNATyr deacylase from Escherichia coli. J. Biol. Chem. 274, 19109–19114 (1996).

    Article  Google Scholar 

  12. Bruckner, H. & Westhauser, T. Chromatographic determination of L- and D-amino acids in plants. Amino acids 24, 43–55 (2003).

    Article  CAS  Google Scholar 

  13. Kuiper, H.A., Kleter, G.A., Noteborn, H.P.J.M. & Kok, E.J. Assessment of the food safety issues related to genetically modified foods. Plant J. 27, 503–528 (2001).

    Article  CAS  Google Scholar 

  14. Hare, P. & Chua, N.H. Excision of selectable marker genes from transgenic plants. Nat. Biotechnol. 20, 575–580 (2002).

    Article  CAS  Google Scholar 

  15. Gleave, A.P., Mitra, D.S., Mudge, S.R. & Morris, B.A. Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol. Biol. 40, 223–235 (1999).

    Article  CAS  Google Scholar 

  16. Sambrook, J. & Russell, D.W. in Molecular cloning: a laboratory manual, edn. 2, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

    Google Scholar 

  17. Koncz, C. & Schell, J. The promoter of T-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of vector. Mol. Gen. Genet. 204, 383–396 (1986).

    Article  CAS  Google Scholar 

  18. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  Google Scholar 

  19. Murashige, T. & Skoog, F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 310–313 (1962).

    Article  Google Scholar 

  20. Schadewaldt, P., Wendel, U. & Hammen, H.W. Determination of R- and S-3-methyl-2-oxopentanoate enantiomers in human plasma: suitable method for label enrichment analysis. J. Chrom. B. 682, 209–218 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge Margareta Zetherström for help with HPLC analyses and several colleagues for comments on earlier versions of this manuscript. Grants from The Swedish Council for Environment, Agricultural Sciences and Spatial planning, the Kempe foundation, the Wallenberg foundation and Carl Tryggers Stiftelse for financial support to T.N. is gratefully acknowledged.

Author information

Author notes

  1. Magnus Hertzberg

    Present address: SweTree Technologies, Uminova Science Park, PO Box 7981, SE-907 19, Umeå, Sweden

Authors and Affiliations

  1. Department of Forest Genetics and Plant Physiology, Umeå Plant Science Center, SLU, Umeå, SE-901 83, Sweden

    Oskar Erikson & Torgny Näsholm

Authors
  1. Oskar Erikson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Magnus Hertzberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Torgny Näsholm
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Torgny Näsholm.

Ethics declarations

Competing interests

The company SweTree Technologies has filed a patent application on the use of D-amino acid–metabolizing enzymes as selectable markers. O.E., M.H. and T.N. are listed as the inventors on this patent application.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Erikson, O., Hertzberg, M. & Näsholm, T. A conditional marker gene allowing both positive and negative selection in plants. Nat Biotechnol 22, 455–458 (2004). https://doi.org/10.1038/nbt946

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt946

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing