Isolation and characterization of cDNA encoding P-19.5 protein accumulated preferentially at early stage of carrot somatic embryogenesis

Abstract

We found P-19.5 protein that preferentially accumulated in cell clusters and globular embryos at the early stage of carrot somatic embryogenesis. P-19.5 protein was isolated from 9-day-old cell clusters with SDS-PAGE, and then determined the amino acid sequences of a N-terminal peptide and some internal peptides. Using each primer for these peptides, the full-length sequence of cDNA encoding P-19.5 protein was determined by analyzing 3′-RACE and 5′-RACE products. We regard P-19.5 protein as an allergen-like protein because the P-19.5 protein sequence predicted from cDNA was homologous to celery major allergen (75%), carrot major allergen (70–72%), Pimpinella brachycarpa pathogenesis-related protein (71%), and parsley pathogenesis-related protein (63%). We found concomitantly two separate cDNAs ( P-16 and P-19.5 H cDNAs) during analyzing P-19.5 cDNA: P-16 cDNA was cloned and isolated from P-16 protein that was recognized against P-19.5 protein antiserum, and P-19.5 H cDNA that had homology (80.5% amino acid identity) with P-19.5 cDNA was detected during analyzing P-19.5 cDNA. Northern blotting revealed that P-19.5 and P-19.5 H transcripts were highly expressed at early stages of cell cluster and globular embryos, whereas P-16 transcript was invariably expressed at all stages from cell clusters to plantlets.