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Analysis of seed storage proteins in transgenic wheat and barley using electrophoresis.
Project Code: G02002;
Salvo G H, Travella S, Bilham L J, Harwood W A, Snape J W (2004) The distribution of transgene insertion sites in barley determined by physical and genetic mapping. Genetics, 167 1371-1379.
John Innes Centre
The seed storage proteins of wheat and barley form an important component of many foods and are responsible for the properties that make wheat and barley grain so important in our diet. Seed storage proteins have been extensively analysed using gel- based methods over many years. It was therefore appropriate to see whether this method could be used for screening GM wheat and barley seed for unexpected consequences of transgene insertions that specifically affected this important group of proteins. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) provides a method to size separate sub-units in cereal protein mixtures providing an accurate method for analysis of seed storage proteins. Cereal proteins have been extensively studied using this technique. In objective 01 we have modified the methodology to provide a standard operating procedure using SDS-PAGE to analyse transgenic wheat and barley grain for unexpected consequences of the transgene insertion that affect the seed storage proteins. Storage proteins account for about 50% of the total protein in mature cereal grain (Shewry and Halford 2002). Hordeins are the major barley storage proteins and comprise the prolamin fraction (Vapa and Radovic, 1998). In wheat the major storage proteins are also in the form of prolamins and have been classified into two groups: the glutenins and gliadins. Reduction of glutenins and their separation by SDS-PAGE indicate that they are comprised of high molecular weight (HMW) and low molecular weight (LMW) subunits (Payne 1987). In the current study to establish methodology for the assessment transgenic wheat and barley grain for unintended effects of transgene insertion affecting the seed storage proteins, transgenic wheat and barley lines were examined alongside null segregant controls (lines derived from the same transformation event that have segregated without the transgene) and non-transformed seed controls. We report on the variation seen within the seed storage proteins that can be attributed to the in vitro culture and transformation process and that attributed specifically to the transgene insertion.
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