Molecular cloning and characterization of nitrogen source responsive GS1 gene from melon

Abstract

A full-length cDNA clone encoding cytosolic glutamine synthetase (GS1; EC 6.3.1.2) was isolated from melon (Cucumis melo L.) for the first time by RT-PCR and RACE approach. The clone, designated as M-GS1 (accession No. DQ851867), contains 1494 nucleotides with an open reading frame (ORF) of 1068 nucleotides. The deduced 356 amino acid sequence showed high similarity with previously reported GS1s from various plant species. Sequence analysis revealed that the predicted protein contains a GS β-Grasp domain, a GS catalytic domain, and the main conserved motifs characteristic of a plant GS1. The phylogenetic analysis displayed that M-GS1 is related most closely to the GS1 from Datisca glomerata. Southern blot analysis indicated that M-GS1 belongs to a small gene family of 2 or 3 members. M-GS1 was expressed in all plant tissues without evident tissue specificity, but with different patterns when the melon plants were fed in hydroponic culture with different forms and concentration of nitrogen. Ammonium dramatically enhanced the contents of M-GS1 transcripts in all tested tissues, while nitrate stimulated M-GS1 transcription only in the roots and leaves, but not in the stems; glutamate, however, depressed M-GS1 transcripts in the roots, but resulted in no significant change to the levels of M-GS1 transcripts in the stems and leaves. Moreover, the same effects were observed at the GS enzyme activity level. These results indicated that melons respond to changes of N nutrition by regulating M-GS1 expression.