Isolation, characterization, and developmental regulation of 2S seed storage protein gene in Pseudotsuga menziesii (douglas fir)
Date
1996
Authors
Machander, David
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Abstract
Studies on the developmental regulation and characterization of genes from Douglas fir were undertaken. A clone (p900) was isolated from a cDNA library prepared from poly A+ mRNA isolated from the mid-stage of Douglas fir embryogenesis. Northern hybridization studies suggested the cDNA to be a full length clone. The gene is clearly developmentally regulated at the transcriptional level rn both the megagametophyte and slightly differently (temporally) in the zygotic embryo. Sequencing data confirmed the clone to be a 2S albumin, yet a unique isoform. Nucleotide sequence alignments showed an overall similarity to Picea glauca albumin and Pinus strobus albumins 1, 2, and 3 of 48.8%. An open reading frame of 140 amino acids encoding a 15.8 kDa preprotein precursor was seen from the predicted amino acid sequence data. A signal peptide of 21 aa was present as predicted by signal peptide cleavage site rules and confirmed by hydropathy plotting. The protein is predicted to have a lack of N-linked glycosylation and is a basic protein. A 30.5% overall amino acid similarity is seen between p900 and other gymnosperm albumins. The protein is high in arginine and glutamine/glutamate at 15.7% and 12.2%, respectively, indicative of the role of albumin as a nitrogen source during seed germination. Additionally, the p900 albumin has a high cysteine content (2.1 % ) and shows a strict conservation in the positioning of these cysteines. Unique features of this isoform include the deletion of two regions where cysteines are typically conserved.