Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers.
The low-molecular-weight (LMW) glutenin subunits are components of the highly cross-linked glutenin polymers that confer viscoelastic properties to gluten and dough. They have both quantitative and qualitative effects on dough quality that may relate to differences in their ability to form the inter-chain disulphide bonds that stabilise the polymers. In order to determine the relationship between dough quality and the amounts and properties of the LMW subunits, we have transformed the pasta wheat cultivars Svevo and Ofanto with three genes encoding proteins, which differ in their numbers or positions of cysteine residues. The transgenes were delivered under control of the high-molecular-weight (HMW) subunit 1Dx5 gene promoter and terminator regions, and the encoded proteins were C-terminally tagged by the introduction of the c-myc epitope. Stable transformants were obtained with both cultivars, and the use of a specific antibody to the c-myc epitope tag allowed the transgene products to be readily detected in the complex mixture of LMW subunits. A range of transgene expression levels was observed. The addition of the epitope tag did not compromise the correct folding of the trangenic subunits and their incorporation into the glutenin polymers. Our results demonstrate that the ability to specifically epitope-tag LMW glutenin transgenes can greatly assist in the elucidation of their individual contributions to the functionality of the complex gluten system.
Rothamsted Research, Harpenden, Herts Al5 2JQ, UK.
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Tosi
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D'Ovidio
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Napier
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Bekes
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Shewry
D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D016259:Genes, myc / Q000235:genetics; D005983:Glutens / Q000031:analogs & derivatives* / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011108:Polymers / Q000378:metabolism; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics; D014908:Triticum / Q000235:genetics*
D011108:Polymers; D005983:Glutens; C037302:glutenin