Molecular cloning and characterisation of two genes encoding asparagine synthetase in barley ( Hordeum vulgare L.)

Abstract

Two different cDNA clones encoding asparagine synthetase (AS: EC 6.3.5.4.) were cloned from barley ( Hordeum vulgare L. cv. Alexis). The corresponding genes were designated HvAS1 (GenBank no AF307145) and HvAS2 (GenBank no AY193714). Chromosomal mapping using wheat–barley addition lines revealed that the HvAS1 gene is located on the long arm of barley chromosome 5, while the HvAS2 gene maps to the short arm of chromosome 3. Both genes are expressed in barley leaves according to RT-PCR analysis but only the HvAS1 gene expression can be detected in roots. Northern blots show no expression of HvAS1 in plants grown under a normal 16 h light/8 h dark cycle but after 10 h of continuous darkness, transcript appears and mRNA accumulates over a 48-h period of dark treatment. In roots, low-level expression of HvAS1 could be detected and the expression level appears to be unaffected by light. A polyclonal antibody was raised against the HvAS1 protein and used in Western blot analysis. The AS protein accumulated during a 48-h period of dark treatment, following the increase in HvAS1 transcript.