Molecular cloning of a gene encoding a seed-specific protein with nutritionally balanced amino acid composition from Amaranthus

Abstract

An albumin with a well-balanced amino acid composition and high levels of the essential amino acids was purified to homogeneity from the mature seeds of Amaranthus hypochondriacus. The amino acid composition of this protein is comparable to the World Health Organization recommended values for a highly nutritional protein. The protein is a 35-kDa monomer with four isoforms that can be separated by chromatofocusing. Antibodies raised against one of the isoforms, AmA1, cross-reacted with the other three isoforms. Affinity-purified AmA1 antibodies were used to isolate cDNA clones from a developing-seed expression library. The six immunopositive recombinants obtained were found to be related. The cDNA of the largest clone (1.2 kilobases) has a single major open reading frame corresponding to a 304-amino acid polypeptide. The clone was confirmed by hybrid-selected translation and immunoprecipitation. The size of the immunoprecipitated product was identical to the mature protein. Analysis of RNA and protein in developing seeds showed that AmA1 is synthesized during early embryogenesis, reaching a maximum by midmaturation. No RNA was detected in 1-day-old seedlings although the protein showed delayed breakdown on germination. Expression of the AmA1 gene was found to be seed-specific, as no protein or RNA was detected in other plant tissues.