TY - JOUR
T1 - Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation
AU - Ebmeier, A.
AU - Allison, L.
AU - Cerutti, H.
AU - Clemente, T.
N1 - Funding Information:
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.
PY - 2004/3
Y1 - 2004/3
N2 - The initial step in the synthesis of isoleucine (I1e) 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 I1e feedback inhibition result in intracellular accumulation of I1e. 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.
AB - The initial step in the synthesis of isoleucine (I1e) 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 I1e feedback inhibition result in intracellular accumulation of I1e. 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.
KW - Agrobacterium
KW - O-Methyl threonine
KW - Threonine dehydratase
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U2 - 10.1007/s00425-003-1129-x
DO - 10.1007/s00425-003-1129-x
M3 - Article
C2 - 14673650
AN - SCOPUS:1642410863
VL - 218
SP - 751
EP - 758
JO - Planta
JF - Planta
SN - 0032-0935
IS - 5
ER -