Molecular cloning and characterization of a legumin-like storage protein cDNA of Douglas fir seeds.

A cDNA library was made from poly(A)+ RNA isolated from developing Douglas fir (Pseudotsuga menziesii) embryo and megagametophytic tissue, and the cDNA clones were identified by immunoscreening with polyclonal antiserum against the crystalloid storage protein complex of Douglas fir. The nucleotide sequence of the longest cDNA insert (DF1) was analysed. The amino acid sequence derived from the DNA sequence verified its identity as a legumin-like storage protein (pseudotsugin) and confirmed that the protein is synthesized as a precursor similar to the 11-12S storage globulins. The transcripts corresponding to cDNA insert DF1 were abundant in the early- to mid-stages of embryogenesis in the diploid embryonic axes as well as in the haploid megagametophytic tissue. The deduced amino acid sequence of pseudotsugin consists of a 29 amino acid N-terminal signal peptide preceding the acidic polypeptide region (286 amino acids) and the subsequent basic polypeptide region (212 amino acids). The site for post-transcriptional cleavage of the precursor polypeptide to make the A and B polypeptides is localized between asparagine -315 and glycine -316 and is highly conserved between angiosperms and gymnosperms. The deduced amino acid sequence for the DF1 cDNA clone reveals that pseudotsugin is rich in arginine, glutamic acid and serine and is low in cysteine, methionine and lysine. Comparison of the deduced amino acid sequence of Douglas fir pseudotsugin shows between 29-38.5% identity with angiosperm species, 63% identity with interior spruce, and 60% identity with eastern white pine.