Cloning of Mannose 6-phosphate Reductase from Celery.

Abstract

In celery, photosynthetic carbon partitioning between mannitol and sucrose is highly dependent on developmental (leaf age) and environmental (salt stress) factors. Mannose 6-phosphate reductase (M6PR) mediates a key step in mannitol biosynthesis and may regulate partitioning between sucrose and mannitol. We have constructed a cDNA library and have isolated M6PR-specific clones. Before library construction, poly(A)+ RNA, extracted from newly fully expanded leaves, was translated in vitro. A single polypeptide (35.1 kD), immunoprecipitated with M6PR-specific antisera, accounted for ≈5% of the total 35S incorporated into TCA-precipitated products. Parity between the molecular masses of the immunoprecipitated product and authentic M6PR indicated minimal posttranslational modification. The unidirectional primary library, constructed in UniZap XR vector (Stratagene), consisted of 1.53 million plaque forming-units (pfus) of which <0.4% were nonrecombinant, as estimated by “blue/white”' screening. After a single amplification, ≈0.14% of the 200,000 pfus screened with M6PR-specific antisera were identified as putative M6PR clones. Following two further rounds of screening and in vivo excision of the pBluescript phagemids their identity as full length M6PR clones was confirmed as follows: 1) IPTG-induced expression of M6PR activity in crude extracts; 2) IPTG-induced expression of a polypeptide that specifically interacted with M6PR antisera and with identical mobility (on SDS gels) to authentic M6PR; 3) 100% sequence homology to an internal peptide from a tryptic digest of purified M6PR. Based on these criteria, we conclude that we successfully cloned M6PR. The sequence is similar to several reductases from both plants and animals including an aldose 6-phosphate reductase from apple. Supported by USDA-NRI grant 940-1439.