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

Ebmeier, A.; Allison, Lesley; Cerutti, Heriberto; Clemente, Thomas E.

doi:10.1007/s00425-003-1129-x

Cited by 20 publications

(8 citation statements)

References 30 publications

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“…These observations have clearly shown that the lyr gene had neither deleterious nor pleiotropic effects on the transgenic plants. In contrast, abnormal morphological phenotypes were commonly observed among transformants selected using ilvA and kn1 or ipt genes as positive selectable markers (Ebmeier et al 2004;Luo et al 2006).…”

Section: Discussionmentioning

confidence: 95%

“…Therefore, it may be possible that further optimization of culture conditions would considerably increase the transformation frequency. In some previous studies demonstrating the utility of new genes as efficient selectable markers in tobacco, transformation frequencies of 13% for xylA gene, almost 20% for ipt gene, 0.7-7.4% for ilvA gene and 19 and 40% for Atwbc19 gene under two different selection conditions had been reported (Haldrup et al 1998;Kunkel et al 1999;Ebmeier et al 2004;Mentewab and Stewart 2005). Both Values represent mean ± SD (n = 10) and are statistically not significant at P B 0.05 a Days are given relative to date of sowing, including a 2-day stratification at 4°C to synchronize seed germination transgenic Arabidopsis and tobacco plants selected on L-lysine exhibited normal growth characteristics as that of wild-type plants.…”

Section: Discussionmentioning

confidence: 99%

“…This phenomenon was successfully demonstrated by employing dao1 and dsdA genes encoding D-amino acid oxidase and D-serine dehydratase, respectively as novel selectable markers for the identification of Arabidopsis transformants (Erikson et al 2004(Erikson et al , 2005. Ebmeier et al (2004) reported the use of ilvA gene, encoding threonine deaminase, as an efficient selectable marker in tobacco using L-O-methylthreonine as the selection agent. These marker genes such as dsdA and ilvA are derived from pathogenic bacterial species, and the incorporation of such genes into foods can aggravate issues of social acceptance.…”

Section: Introductionmentioning

confidence: 99%

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Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

et al. 2009

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A non-antibiotic based selection system using L-lysine as selection agent and the lysine racemase (lyr) as selectable marker gene for plant transformation was established in this study. L-lysine was toxic to plants, and converted by Lyr into D-lysine which would subsequently be used by the transgenic plants as nitrogen source. Transgenic tobacco and Arabidopsis plants were successfully recovered on L-lysine medium at efficiencies of 23 and 2.4%, respectively. Phenotypic characterization of transgenic plants clearly revealed the expression of normal growth and developmental characteristics as that of wild-type plants, suggesting no pleiotropic effects associated with the lyr gene. The specific activity of Lyr in transgenic tobacco plants selected on L: -lysine ranged from 0.77 to 1.06 mU/mg protein, whereas no activity was virtually detectable in the wild-type plants. In addition, the composition of the free amino acids, except aspartic acid, was not affected by the expression of the lyr gene in the transgenic tobacco plants suggesting very limited interference with endogenous amino acid metabolism. Interestingly, our findings also suggested that the plant aspartate kinases may possess an ability to distinguish the enantiomers of lysine for feedback regulation. To our knowledge, this is the first report to demonstrate that the lysine racemase selectable marker system is novel, less controversial and inexpensive than the traditional selection systems.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

et al. 2009

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“…The ligand binds at the interface between the ACT domains. 23 − 25 In this work, the MBAP method has been used to predict the indirect-binding sites of TD and potential mutations to relieve allosteric regulation. Then, the predicted mutations have been verified by experimental tests.…”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics-Based Allosteric Prediction Method to Design Key Residues in Threonine Dehydrogenase for Amino-Acid Production

Sun

Zhu

et al. 2021

ACS Omega

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Allosteric proteins are considered as one of the most critical targets to design cell factories via synthetic biology approaches. Here, we proposed a molecular dynamics-based allosteric prediction method (MBAP) to screen indirect-binding sites and potential mutations for protein re-engineering. Using this MBAP method, we have predicted new sites to relieve the allosteric regulation of threonine dehydrogenase (TD) by isoleucine. An obtained mutation P441L has been verified with the ability to significantly reduce the allosteric regulation of TD in vitro assays and with the fermentation application in vivo for amino-acid production. These findings have proved the MBAP method as an effective and efficient predicting tool to find new positions of the allosteric enzymes, thus opening a new path to constructing cell factories in synthetic biology.

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“…Compared with the existing selection marker genes, the selection of novel selection marker genes is an effective method to eliminate their potential risks. According to the mechanism of action of the safety marker developed and the type of agent selected, it can be divided into amino acid metabolism related genes [11,12], hormone related genes [13,14], sugar metabolism related genes [15], stress resistance related genes [16], and fluorescent protein genes [17,18,19]. The use of these selection markers reduces the potential risks of transgenic plants.…”

Section: Introductionmentioning

confidence: 99%

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Jin

Guo

et al. 2019

Genes

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Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

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

Cited by 20 publications

References 30 publications

Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

Molecular Dynamics-Based Allosteric Prediction Method to Design Key Residues in Threonine Dehydrogenase for Amino-Acid Production

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

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