title: A point mutation in the Medicago sativa GSA gene provides a novel, efficient, selectable marker for plant genetic engineering.
abstract: Bacterial selectable marker genes (SMG) conferring antibiotic resistance are valuable tools in plant genetic engineering, but public concern and regulatory requirements have stimulated the development of alternative selection systems. We have previously demonstrated that a mutated Synechococcus elongatus HemL gene encoding glutamate 1-semialdehyde aminotransferase (GSA) is an efficient SMG in alfalfa. In fact, GSA is irreversibly inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid), but the mutated enzyme is gabaculine insensitive. With the aim to develop a plant derived SMG, we cloned and sequenced the Medicago sativa GSA cDNA and reproduced one of the two mutations associated with gabaculine resistance in Synechococcus, a transversion resulting in a methionine to isoleucine (M→I) substitution. This mutated gene was assessed as a SMG in tobacco and alfalfa Agrobacterium transformation, in comparison with the wild type gene. In tobacco, about 43% of the leaf explants produced green shoots, whereas in alfalfa 47% of the explants produced green embryos in the presence of 30 μM gabaculine when the M→I GSA was introduced. Escapes were absent in tobacco and only 6% in alfalfa. No effect on the plant phenotype was noticed. We propose this new SMG as a widely acceptable alternative to those currently used.
authors.affiliation: Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy. nicoletta.ferradini@gmail.com
authors.forename: N
authors.initials: N
authors.lastname: Ferradini
authors.forename: A
authors.initials: A
authors.lastname: Nicolia
authors.forename: S
authors.initials: S
authors.lastname: Capomaccio
authors.forename: F
authors.initials: F
authors.lastname: Veronesi
authors.forename: D
authors.initials: D
authors.lastname: Rosellini
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D015415:Biomarkers; D003001:Cloning, Molecular; D003509:Cyclohexanecarboxylic Acids; D018076:DNA, Complementary; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005818:Genetic Engineering; D019751:Intramolecular Transferases / Q000235:genetics*; D000455:Medicago sativa / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D017354:Point Mutation; D046940:Synechococcus / Q000235:genetics
chemical_list: D015415:Biomarkers; D003509:Cyclohexanecarboxylic Acids; D018076:DNA, Complementary; D010940:Plant Proteins; C012987:gabaculine; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase