Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo

Abstract

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequences on two cyanogen bromide fragments were determined. An oligonucleotide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3-5-day old seedling hypocotyls, in lambda ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539-1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7-15 per haploid genome).