Abstract
In this study, the d-serine ammonia lyase (dsdA) gene from Escherichia coli was evaluated as a selectable marker for maize transformation. Plants are incapable of utilizing the D-form of most amino acids, and d-serine has recently been demonstrated to be phytoinhibitory to plant growth. d-Serine ammonia lyase detoxifies d-serine via a substrate-specific reaction to pyruvate, ammonia, and water. d-Serine inhibits germination of isolated maize immature embryos and growth of embryogenic callus from wild-type plants at concentrations about approx. 2–15 mM. Transgenic plants were recovered in the presence of d-serine in tissue culture media with dsdA as the selection marker at efficiencies comparable to using a mutated acetohydroxy acid synthase selection marker gene and selection in the presence of imidazolinone herbicides. Immature embryos infected with an Agrobacterium strain containing an acetohydroxy acid synthase gene construct without dsdA did not yield any transgenic events on the selection medium with 10 mM d-serine, indicating that d-serine provided selection tight enough to prevent escapes. Molecular analysis confirmed the integration of the dsdA gene into the genome of the transgenic plants. No adverse phenotypes were observed in the greenhouse, and expression of the dsdA marker had no affect on agronomic characteristics or grain yield in multi-location field trials. Seed compositional analysis demonstrated no significant differences in the contents of seed protein, starch, fatty acids, fiber, phytic acid, and free amino acids between transgenic and non-transgenic control plants. These data indicate that the dsdA gene is properly expressed in maize and the d-serine ammonia lyase (DSDA) enzyme functions appropriately to metabolize d-serine during in vitro selection. Preliminary safety assessments indicated that no adverse affects would be expected if humans were exposed to the DSDA protein in the diet from an allergenicity or toxicity perspective. The dsdA gene in combination with phytoinhibitory levels of d-serine represents a new and effective selectable marker system for maize transformation.
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References
Bruckner H.; Westhauser T. Chromatographic determination of l- and d-amino acids in plants. Amino Acids 24: 43–55; 2003.
Carlson J. E.; Tulsieram L. K.; Glaubitz J. C.; Luk V. W. K.; Kauffeldt C.; Rutledge R. Segregation of random amplified DNA markers in F1 progeny of conifers. Theor. Appl. Genet. 83: 194–200; 1991.
Cosloy S. D.; McFall E. Metabolism of d-serine in Escherichia coli K-12: Metabolism of growth inhibition. J. Bacteriol. 114: 685–694; 1973.
De Block M.; Botterman J.; Vandewiele M.; Dockx J.; Thoen C.; Gossele V.; Rao Movva N.; Thompson C.; Van Montague M.; Leemans J. Engineering herbicide resistance in plants by expression of a detoxifiying enzyme. EMBO J. 6: 2513–2518; 1987.
Delaney B.; Astwood J. D.; Cunny H.; EichenConn R.; Herouet-Guicheney C.; MacIntosh S.; Meyer L. S.; Privalle L.; Goa Y.; Mattsson J.; Levine M. Evaluation of protein safety in the context of agricultural biotechnology. Food Chem. Toxicol. 46: S71–S97; 2008.
Erikson O.; Hertzberg M.; Torgny N. A conditional marker gene allowing both positive and negative selection in plants. Nat. Biotechnol. 22: 455–458; 2004.
Erikson O.; Hertzberg M.; Torgny N. The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation. Plant Mol. Biol. 57: 425–433; 2005.
Fraley R. T.; Rogers S. B.; Horsch R. B. Use of a chimeric gene to confer antibiotic resistance to plant cells. Advances in gene technology: Molecular genetics of plants and animals. Miami Winter Symp. 20: 211–221; 1983.
Hiei Y.; Ohta S.; Komari T.; Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of T-DNA. Plant J. 6: 271–282; 1994.
Ishida Y.; Saito H.; Ohta S.; Hiei Y.; Komari T.; Kumashiro T. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat. Biotechnol. 14: 745–750; 1996.
Komari T.; Hiei Y.; Saito Y.; Murai N.; Kumashiro T. Vectors carrying two separate T-DNAs for co-transformation for higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J. 10: 165–174; 1996.
Maas W. K.; Mass R.; McFall E. d-Serine deaminase is a stringent selective marker in genetic crosses. J. Bacteriol. 177: 459–461; 1995.
Marceau M.; McFall E.; Lewis S. D.; Schafer J. A. d-Serine dehydratase from Escherichia coli. J. Biol. Chem. 263: 16296–16933; 1988.
Miki B.; McHugh S. Selectable marker genes in transgenic plants: Applications, alternatives and biosafety. J. Biotechnol. 107: 193–232; 2004.
Murashige T.; Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 310–313; 1962.
Nasholm T.; Sandberg G.; Ericsson A. Quantitative analysis of amino acids in conifer tissues by high-performance liquid chromatography and fluorescence detection of their 9-fluorenlmethyl chloroformate derivatives. J. Chromatogr. 396: 225–236; 1987.
Peng J, Mrabet P, Bagley C, Wang X, Mankin L, Singh B, Lai F-M, Mei K, Zhang Z. Improved methods for the production of stably transformed, fertile Zea mays plants. WO2006/136596; 2006
Sambrook J.; Fritsch E. F.; Maniatis T. Molecular cloning—A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press, New York; 1989.
Szamosi I.; Shaner D. L.; Singh B. K. Identification and characterization of a biodegradative form of threonine dehydratase in senescing tomato (Lycopersicon esculentum) leaf. Plant Physiol. 101: 999–1004; 1993.
Taylor S. L. Chemistry and detection of food allergens. Food Technol. 46: 146–152; 1992.
Thomas K.; Aalbers M.; Bannon G. A.; Bartels M.; Dearman R. J.; Esdaile D. J.; Fu T. J.; Glatt C. M.; Hadfield N.; Hatzos C.; Hefle S. L.; Heylings J. R.; Goodman R. E.; Henry B.; Herouet C.; Holsapple M.; Ladics G. S.; Landry T. D.; MacIntosh S. C.; Rice E. A.; Privalle L. S.; Steiner H. Y.; Teshima R.; van Ree R.; Woolhiser M.; Zawodny J. A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. Regul. Toxicol. Pharmacol. 39: 87–98; 2004.
U.S. Pharmacopoeia (2000) The National Formulary. USP XIV, NF XIX, US Pharmacopoeia Convention, Inc., Mack Printing Col, Easton, PA, USA: 2235
Vorachek-Warren M. K.; McCusker J. H. DsdA (d-serine deaminase): A new heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae. Yeast 21: 163–171; 2004.
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The authors would like to thank Hongmei Jia for her statistical analysis of the experimental data.
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Lai, FM., Privalle, L., Mei, K. et al. Evaluation of the E. coli d-serine ammonia lyase gene (Ec. dsdA) for use as a selectable marker in maize transformation. In Vitro Cell.Dev.Biol.-Plant 47, 467–479 (2011). https://doi.org/10.1007/s11627-011-9351-x
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DOI: https://doi.org/10.1007/s11627-011-9351-x