Molecular cloning and characterization of a cytosolic glutamine synthetase gene, a fiber strength-associated gene in cotton

Abstract

An expressed sequence tag encoding glutamine synthetase (GS) was identified by microarray-based hybridization using fiber mRNAs of allotetraploid Gossypium hirsutum, 7235, a super quality property germplasm line, and TM-1, a genetic standard in G. hirsutum. Northern-blot analysis verified transcript accumulation differences in 8 DPA fibers (including ovules) in the two varieties. The full-length cDNA encoding GS in 7235 was isolated and named GhGS. Sequence analysis revealed that the GhGS was similar to cytosolic GS. Southern-blot analysis showed that tetraploid cotton contained at least one copy of the A sub-genome and the D sub-genome. Genomic GhGS sequences were subsequently isolated from different varieties, TM-1, 7235 and two diploid progenitor cottons, G. herbaceum (A-genome) and G. raimondii (D-genome). Molecular mapping and single-marker analysis revealed that the GhGS was significantly correlated with fiber strength and was mapped to chromosome D7. Additionally, GS activities and total protein of the ovules and the fibers were assayed. The results showed a significantly higher GS activity in 7235 seeds compared to TM-1 seeds at 5 and 8 DPA. Also significant differences were found in total protein content and seed weight at 11 DPA. This suggested that GS promoted the seed-forming process by providing N. On the other hand, in fibers, GS activity and total protein assay indicated a lower total GS activity and longer fiber elongation period in 7235. These results suggest that the respective roles of the GS in ovules and fibers do not completely overlap.