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Proceedings of the 53rd Italian Society of Agricultural Genetics Annual Congress
Torino, Italy – 16/19 September, 2009
ISBN 978-88-900622-9-2
Poster Abstract – 7.50
MEDICAGO SATIVA GSA-AT, A NOVEL, PLANT-DERIVED SELECTABLE
MARKER GENE FOR GENETIC ENGINEERING
FERRADINI N., NICOLIA A., FUSCO C., CAPOMACCIO S., VERONESI F., ROSELLINI D.
Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Borgo XX Giugno 74,
06121 Perugia, Italy
selectable marker genes, GSA-AT, gabaculine, Nicotiana tabacum, Medicago sativa
The use of selectable marker genes (SMG) of bacterial origin conferring antibiotic or
herbicide resistance has been a valuable tool in plant genetic engineering for many years. Consumer
concerns and regulatory requirements have stimulated the development of alternative selection
systems. In previous experiments, we have estimated that the efficiency of standard marker-free
techniques is at present too poor for routine use in alfalfa.
We have previously demonstrated that a mutated form of the Synechococcus elongatus hemL
gene encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) is efficient a SMG in alfalfa.
The enzyme GSA-AT catalyses the conversion of glutamate-1-semialdehyde into aminolevulinic
acid, a step in the synthesis of tetrapyrrole compounds, including chlorophyll. GSA-AT is
irreversibly inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). The mutated bacterial gene
has a point mutation resulting in the substitution of a methyonine with an isoleucine in the catalytic
site of the enzyme, and a three aminoacid deletion close to the N terminus.
With the aim to develop a plant-derived, non-antibiotic, safe, SMG, we cloned and sequenced
the Medicago sativa (GSA-AT) cDNA. Then we point-mutated the gene by PCR, so to reproduce
the bacterial methyonine to isoleucine substitution.
This mutated GSA-AT gene (MsGSA-gr) was assessed for the ability to confer gabaculine
resistance in Nicotiana tabacum and Medicago sativa transformation via Agrobacterium
tumefaciens.
Two transformation experiments were performed for both species. In tobacco, 46,5% and
40,3% of the leaf explants produced green shoots in the presence of 30 µM gabaculine. In alfalfa,
the observed percentages have turned out higher: 92,3 % of the explants produced green embryos.
Moreover our preliminary data indicate the complete absence of escapes in both species.
The very good results achieved with tobacco and alfalfa transformation suggests that this new
SMG can be applied with success to other plant species. Because of the specificity of the GSA-AT
enzyme and the plant origin of the marker, we think that it could be considered a favourable
alternative to currently used SMGs both for the risk assessment process and for public acceptance.