A gene for legumin: a major storage protein of pisum sativum L.

Abstract

A gene coding for legumin, a major storage protein of pea, was isolated from a library of pea DNA, and partially sequenced. The library was constructed by partially digesting pea leaf DNA with Sau 3A, and ligating 15 - 20 Kb size fractions into the Bam H1 cleaved arms of the vector λ L47. Ribosomal clones were isolated from the library at a frequency which indicated that the sequence representation was near complete. Screening of the library for sequences encoding legumin was performed by using legumin complementary DNA. The coding sequence of this gene was localised using restriction enzyme and hybridisation analysis, to a 2.7 kb fragment within the 15 Kb genomic insert in the phage vector. This 2.7 Kb fragment was subcloned into the plasmid vector pUC 8, and analysed by restriction enzyme mapping and sequencing. This showed the presence of three small intervening sequences within the legumin coding region. Sequence data obtained for two of the intervening sequences indicated that they were rich in adenine and thymine nucleotides, and that the 5' terminus of both introns began with the dinucleotide GT. Hybridisation experiments using this gene to probe for homologous sequences in pea leaf DNA detected 7 hybridising fragments, suggesting the existence of more genes than were originally thought to comprise the legumin gene family.