Identification of novel α-gliadin genes

Abstract

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.

An Overexpressed Q Allele Leads to Increased Spike Density and Improved Processing Quality in Common Wheat (Triticum aestivum)

“…Seed storage proteins were extracted from 20 mg whole wheat flour and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Qi et al (2011) .…”

An overexpressedQallele leads to increased spike density and improved processing quality

“…Seed storage proteins were extracted from 20 mg seed powder and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Qi et al (2011).…”

Identification of a Group of Novel γ-Gliadin Genes

“…We have characterized ten novel α-gliadin genes, which resulted from illegitimate recombination between the two polyglutamine regions (Qi et al 2011). The following similarities exist between the novel γ-gliadin genes and novel α-gliadin genes: the central non-repetitive domain/unique domain containing most of the cysteine residues was lost; the regions enriched with glutamine (including the repetitive domain, polyglutamine region of α-gliadins and glutamine-rich region of γ-gliadins) were partially or completely lost; their remaining sequences were identical to the typical γ-and α-gliadin genes respectively; they are chain extenders in in vitro assay; their molecular weights were remarkably smaller than those of typical gliadins.…”

“…Growth of bacteria in Luria-Bertani media and purification of over expressed gliadins from E. coli were performed following the protocol of Qi et al (2011). The in vitro expressed Gli-ng1 were then dissolved in a reducing (Mixture A: 62.5 mmol L -1 Tris-HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS (sodium dodecyl sulfate), 0.002% (w/v) bromophenol blue, 1.5% (w/v) DTT (dithiothreitol)) or non-reducing solution (Mixture B:…”

“…Method 2: Milled seeds (0.1 g) were suspended in 1.2 mL 70% ethanol, and then incubated at 60º C for 2 h. Seed debris was removed by centrifugation (10 min at 21000×g). Alcohol soluble seed extracts were extracted from the supernatant following the protocol of (Qi et al 2011), and dissolved in mixture A (reducing solution) and B…”