A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids.
A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.
Institute of Biosciences and Bioresources, Research Division of Perugia, National Research Council (CNR), via della Madonna Alta 130, 06128, Perugia, Italy.
Michele
M
Bellucci
Institute of Biosciences and Bioresources, Research Division of Perugia, National Research Council (CNR), via della Madonna Alta 130, 06128, Perugia, Italy.
Francesca
F
De Marchis
Department of Agricultural Food and Environmental Sciences, University of Perugia, Borgo XX Giugno 74, 06121, Perugia, Italy.
Nicoletta
N
Ferradini
Institute of Biosciences and Bioresources, Research Division of Perugia, National Research Council (CNR), via della Madonna Alta 130, 06128, Perugia, Italy.
Andrea
A
Pompa
Department of Agricultural Food and Environmental Sciences, University of Perugia, Borgo XX Giugno 74, 06121, Perugia, Italy.
Fabio
F
Veronesi
Department of Agricultural Food and Environmental Sciences, University of Perugia, Borgo XX Giugno 74, 06121, Perugia, Italy. daniele.rosellini@unipg.it.
Daniele
D
Rosellini
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005819:Genetic Markers / Q000235:genetics; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D000455:Medicago sativa / Q000235:genetics / Q000502:physiology; D009154:Mutation; D010788:Photosynthesis; D030821:Plants, Genetically Modified; D018087:Plastids / Q000201:enzymology; D046940:Synechococcus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D014026:Nicotiana / Q000235:genetics / Q000502:physiology; D014908:Triticum / Q000235:genetics / Q000502:physiology
D001426:Bacterial Proteins; D003509:Cyclohexanecarboxylic Acids; D004791:Enzyme Inhibitors; D005819:Genetic Markers; C012987:gabaculine; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase
Chloroplast; Glutamate 1-semialdehyde aminotransferase; In vitro selection; Transgenic plants; Transplastomic plants