Differential accumulation of four phaseolin glycoforms in transgenic tobacco |
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Authors: | Mauricio M Bustos Fatma A Kalkan Kathryn A VandenBosch Timothy C Hall |
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Institution: | (1) Department of Biology, Texas A & M University, 77843-3258 College Station, TX, USA |
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Abstract: | An intron-less phaseolin gene 15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type ( wti–) and mutant phaseolin glycoforms ( dgly
1, dgly
2 and dgly
1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly
1 and dgly
2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly
1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp
base pair(s)
- DAF
days after flowering
- GUS
-glucuronidase
- kb
kilobase
- kDa
kilodalton |
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Keywords: | French bean (Phaseolus vulgaris) N-glycosylation phaseolin plant introns protein stability seed storage proteins |
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