Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery.

摘要

Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, Western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis and M6PR activity.