EXPRESSION OF GLUTAMINE SYNTHETASE (GS) GENES IN DURUM WHEAT CULTIVARS CHARACTERIZED BY A DIFFERENT GRAIN PROTEIN CONTENT

Glutamine synthetase (GS) is a key enzyme for nitrogen (N) assimilation in plants, which catalyses the ATP-dependent condensation of ammonium and glutamate into glutamine, the principal precursor for the synthesis of most nitrogenous cellular compounds. Glutamine synthetase exists in different isoforms classified into groups according to their localization within the cell: the cytosolic form, GS1, and the chloroplastic form, GS2. GS1 is responsible for assimilating the ammonium produced by reduction of nitrate in roots, and for synthesizing Gln for the transport of N between different organs, while the major function of GS2 is to reassimilate ammonia endogenously released by photorespiration. The goal of the present study was to assess a specific and reliable protocol of RT-real time PCR for the study of the GS genes differential expression in two durum wheat cultivars Ciccio and Svevo, characterized respectively by high and low kernel protein content. The expression study was conducted on several tissues and variable developmental stages. In particular, total RNA was extracted and cDNA synthesized from leaves and roots collected from ten wheat plants at different phonological stages, from branching to grain filling. In order to optimize the PCR reaction conditions, a set of six housekeeping genes represented by the selected sequences of Actin, α-tubulin, TEF-1α, ADP-RF, RLI and CDC, were assessed by preliminary qRT-PCR assays. Some discrepancies were observed in the ranking of the candidate reference genes, and three of them (RLI, CDC and ADP-RF), which appeared more effective, were chosen for the study of GS genes. Real Time PCR conducted for plastidic GS2-A2 and GS2-B2 genes, and for cytosolic GSe1 and GSe2 genes, showed a different expression pattern in the various developmental stages for both cvs. Ciccio and Svevo.