cDNA clones for Brassica napus seed storage proteins: evidence from nucleotide sequence analysis that both subunits...

Napins are a family of small, basic storage proteins synthesized in Brassica napus (rapeseed) embryos during seed maturation. Cultured embryos also synthesize napins but require exogenous abscisic acid (ABA) to maintain high accumulation rates. We synthesized cDNA from total RNA of embryos cultured on a medium containing ABA, and cloned it into the Pst1 site of pBR322. Two clones containing napin cDNA sequences selected by differential colony hybridization using [32P]cDNA probes from embryos grown with or without ABA were analyzed. These clones, pN1 (insert size = 583 bp) and pN2 (insert size = 739 bp), contained cDNA from two different napin mRNAs. The mRNAs to which they hybridized were found to encode a 21,000-dalton polypeptide that was immunoprecipitated by antibodies to mature napin (subunits of 9,000 and 4,000 daltons). The cDNA clones hybridized to an 850-base mRNA. Nucleotide sequencing demonstrated 95% homology between pN1 and pN2 cDNA inserts and predicted a precursor polypeptide of 178 amino acids, consistent with the 21,000 dalton in vitro translation product. Comparison of the deduced amino acid sequence with published amino acid compositions of mature napin subunits suggests that both the large and the small subunits are present in one precursor polypeptide, and that other regions of the precursor are removed during processing.