Grain protein content (GPC) of wheat is directly related to the nutritional and technological value of the derived products. Several studies have attested the key role of the glutamine synthetase enzyme (GS) in plant nitrogen metabolism, as the responsible of the first step of ammonium assimilation and transformation into glutamine, an essential compound in the amino acidbiosynthetic pathway. GS enzyme exists in multiple forms, with the chloroplastic isozyme encoded by one gene (GS2) and the cytosolic one encoded by 3-5 different genes, depending on the species. Both GS isozymes are developmentally regulated in leaves, and have different metabolic roles and chromosomal locations. The goal of the present work was to isolate and characterize the GSe1 and GSe2 genes for the enzymatic cytosolic isoforms in two durum wheat cultivars, Ciccio and Svevo, respectively characterized by a high and a low grain protein content, and to localize them on the homoeologous group 4 chromosomes, where QTLs for GPC were detected in several studies. To isolate the complete genomic sequences of glutamine synthetase genes in durum wheat, we used the recently cloned cDNA sequences from hexaploid wheat AY491970 and AY491971, identifying GSe1 and GSe2 isozymes, respectively. The two sequences were blasted against the 454 reads in the Cereal DB database, thus obtaining a contig of 3037 bp for GSe1 and a contig of 2576 bp for GSe2. A set of different primer pairs was specifically designed on the basis of each contig in order to cover the entire genomic sequence. Single PCR fragments were directly purified with EuroGold Cycle Pure Kit and sequenced. The sequence was completed by a Genome Walker approach. The gene structure of GSe1 and GSe2 was predicted for both the two durum wheat cultivars by using the software FGENESH 2.6. Both the GSe genes accounted for a total of 11 exons separated by 10 introns, and have a CDS sequence of 1140 bp for GSe1 and 966 bp for GSe2. The molecular structure appeared very conserved in the two genes, with only slight differences in the last two exons, which were longer in GSe1. Finally, nulli-tetrasomic, di-telosomic and deletion bin lines derived from the hexaploid cv. Chinese Spring were used for physical location of the two genes on 4A and 4B chromosome bins.
ISOLATION AND CHARACTERIZATION OF CYTOSOLIC GLUTAMINE SYNTHETASE (GSe) GENES IN DURUM WHEAT
GIANCASPRO A;
2012-01-01
Abstract
Grain protein content (GPC) of wheat is directly related to the nutritional and technological value of the derived products. Several studies have attested the key role of the glutamine synthetase enzyme (GS) in plant nitrogen metabolism, as the responsible of the first step of ammonium assimilation and transformation into glutamine, an essential compound in the amino acidbiosynthetic pathway. GS enzyme exists in multiple forms, with the chloroplastic isozyme encoded by one gene (GS2) and the cytosolic one encoded by 3-5 different genes, depending on the species. Both GS isozymes are developmentally regulated in leaves, and have different metabolic roles and chromosomal locations. The goal of the present work was to isolate and characterize the GSe1 and GSe2 genes for the enzymatic cytosolic isoforms in two durum wheat cultivars, Ciccio and Svevo, respectively characterized by a high and a low grain protein content, and to localize them on the homoeologous group 4 chromosomes, where QTLs for GPC were detected in several studies. To isolate the complete genomic sequences of glutamine synthetase genes in durum wheat, we used the recently cloned cDNA sequences from hexaploid wheat AY491970 and AY491971, identifying GSe1 and GSe2 isozymes, respectively. The two sequences were blasted against the 454 reads in the Cereal DB database, thus obtaining a contig of 3037 bp for GSe1 and a contig of 2576 bp for GSe2. A set of different primer pairs was specifically designed on the basis of each contig in order to cover the entire genomic sequence. Single PCR fragments were directly purified with EuroGold Cycle Pure Kit and sequenced. The sequence was completed by a Genome Walker approach. The gene structure of GSe1 and GSe2 was predicted for both the two durum wheat cultivars by using the software FGENESH 2.6. Both the GSe genes accounted for a total of 11 exons separated by 10 introns, and have a CDS sequence of 1140 bp for GSe1 and 966 bp for GSe2. The molecular structure appeared very conserved in the two genes, with only slight differences in the last two exons, which were longer in GSe1. Finally, nulli-tetrasomic, di-telosomic and deletion bin lines derived from the hexaploid cv. Chinese Spring were used for physical location of the two genes on 4A and 4B chromosome bins.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
