Abstract
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.
References
Borroto K, DureIII L: The globulin seed storage proteins of flowering plants are derived from two ancestral genes. Plant Mol Biol 8: 113–131 (1987).
Casey R, Domoney C, Ellis N: Legume storage proteins and their genes. Oxford Surv Plant Mol Cell Biol 3: 1–95 (1986).
Chlan CA, Pyle JB, Legocki AB, DureIII L: Developmental biochemistry of cotton seed embryogenesis and germination. XVIII. cDNA and amino acid sequences of members of the storage protein families. Plant Mol Biol 7: 474–489 (1986).
Green MJ, McLeod JK, Misra S: Characterization of Douglas fir protein body composition by SDS-PAGE and electron microscopy. Plant Physiol Biochem 29: 1–7 (1991).
Lutcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GAL: Selection of AUG initiation codons differs in plants and animals. EMBO J 6: 43–48 (1987).
Lycett GW, Croy RRD, Shirsat AH, Boutler D: The complete nucleotide sequence of a legumin gene from pea (Pisum sativum L.). Nucl Acids Res 12: 4493–4506 (1984).
Misra S, Green MJ: Developmental gene expression in conifer embryogenesis and germination. I. Seed proteins and protein body composition of mature embryo and the megagametophyte of white spruce (Picea glauca [Moench] Voss). Plant Sci 68: 163–173 (1990).
Misra S, Green MJ: Developmental gene expression in conifer embryogenesis and germination. II. Crystalloid protein synthesis in the developing embryo and megagametophyte of white spruce (Picea glauca [Moench] Voss). Plant Sci 78: 61–71 (1991).
Misra S, Leal I: Molecular cloning of crystalloid protein cDNAs of Douglas fir. In: Proceedings of the IVth International Workshop on Seeds, (Angers, France), in press.
Pang PP, Pruitt RC, Meyerowitz EM: Molecular cloning, genomic organization, expression and evolution of 12S seed storage protein genes of Arabidopsis thaliana. Plant Mol Biol 11: 805–820 (1988).
Pearson RW, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448 (1988).
Shotwell MA, Larkins BA: The Biochemistry and Molecular Biology of Seed Storage Proteins. In: Marcus A (ed) The Biochemistry of Plants, Vol 15: Molecular Biology, pp. 297–345. Academic Press, New York (1989).
Simon AE, Tenbarge KM, Scofield SR, Finkelstein RR, Crouch ML: Nucleotide sequence of a cDNA clone of Brassica napus 12S storage protein shows homology with legumin from Pisum sativum. Plant Mol Biol 5: 191–201 (1985).
Staswick PE, Hermodson MA, Nielson NC. Identification of the cystines which link the acidic and basic components of the glycinin subunits J. Biol. Chem. 259: 13431–13435 (1984).
VonHeijne G: Patterns of amino acids near signalsequence cleavage sites. Eur J Biochem 133: 17–21 (1983).
Wen L, Huang JK, Johnson BH, Reeck GR: Nucleotide sequence of a cDNA that encodes a rice glutelin. Nucl Acids Res 17: 9490 (1989).
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Leal, I., Misra, S. Molecular cloning and characterization of a legumin-like storage protein cDNA of Douglas fir seeds. Plant Mol Biol 21, 709–715 (1993). https://doi.org/10.1007/BF00014555
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DOI: https://doi.org/10.1007/BF00014555