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Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation

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Abstract

The initial step in the synthesis of isoleucine (Ile) is the conversion of threonine to α-ketobutyrate. This reaction is carried out by threonine deaminase (TD), which is feedback-regulated by Ile. Mutations in TD that manifest insensitivity to Ile feedback inhibition result in intracellular accumulation of Ile. Previous reports have shown that in planta expression of the wild-type Escherichia coli TD, ilvA, or an Ile-insensitive mutant designated ilvA-466, increased cellular concentrations of Ile. A structural analog of Ile, l-O-methylthreonine (OMT), is able to compete effectively with Ile during translation and induce cell death. It has been postulated that OMT could therefore be utilized as an effective selective agent in plant engineering studies. To test this concept, we designed two binary plasmids that harbored an nptII cassette and either the wild-type ilvA or mutant ilvA-466. The ilvA coding sequences were fused to a plastid transit peptide down stream of a modified 35S CaMV promoter. Tobacco transformations were set up implementing a selection protocol based on either kanamycin or OMT. The ilvA gene was effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with OMT as the selection agent. However, the transformation efficiency was substantially lower than that observed with nptII using kanamycin as the selection agent. Moreover, in a subset of the ilvA transformants and in a majority of the ilvA-466 transgenic lines, a severe off-type was observed under greenhouse conditions that correlated with increased levels of expression of the ilvA transgene.

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Abbreviations

ELISA:

enzyme-linked immunosorbent assay

Ile:

isoleucine

OMT:

l-O-methylthreonine

nptII:

neomycin phosphotransferase II

TD:

threonine deaminase

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Acknowledgements

The ilvaA, ilva-466 genes and TD antibodies were kindly provided by Monsanto Company. Gratitude is extended to Tim Mitsky for thoughtful discussions on this project. Pioneer Hi-Bred provided fellowship support for A. Ebmeier. This project was partially supported from monies from the Nebraska Research Initiative and the University of Nebraska’s Center for Biotechnology. This paper is a contribution of the University of Nebraska Agricultural Research Division. This is Journal Series No. 14157.

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Authors and Affiliations

  1. Department of Agronomy & Horticulture, University of Nebraska, Lincoln, NE 68588-0665, USA

    A. Ebmeier & T. Clemente

  2. Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0665, USA

    L. Allison

  3. School of Biological Sciences, University of Nebraska, Lincoln, NE 68588–0665, USA

    H. Cerutti

  4. Center for Biotechnology, University of Nebraska, Lincoln, NE 68588-0665, USA

    T. Clemente

  5. Plant Science Initiative, University of Nebraska, Lincoln, NE 68588-0665, USA

    H. Cerutti & T. Clemente

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  1. A. Ebmeier
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  2. L. Allison
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  4. T. Clemente
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Correspondence to T. Clemente.

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Ebmeier, A., Allison, L., Cerutti, H. et al. Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation. Planta 218, 751–758 (2004). https://doi.org/10.1007/s00425-003-1129-x

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  • DOI: https://doi.org/10.1007/s00425-003-1129-x

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