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

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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.

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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.

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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.

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Correspondence to Torgny Näsholm.

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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.

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

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