Comparative proteomic analysis of two transgenic low-gliadin wheat lines and non-transgenic wheat control

Abstract

Gluten proteins are major determinants of the bread making quality of wheat, but also of important wheat-related disorders, including coeliac disease (CD), and allergies. We carried out a proteomic study using the total grain proteins from two low-gliadin wheat lines, obtained by RNAi, and the untransformed wild type as reference. The impact of silencing on both target and on non-target proteins was evaluated. Because of the great protein complexity, we performed separate analyses of four kernel protein fractions: gliadins and glutenin subunits, and metabolic and CM-like proteins, by using a classical 2D electrophoresis gel based approach followed by RP-HPLC/nESI-MS/MS. As a result of the strong down-regulation of gliadins, the HMW-GS, metabolic and chloroform/methanol soluble proteins were over-accumulated in the transgenic lines, especially in the line D793, which showed the highest silencing of gliadins. Basing on these data, and considering that metabolic proteins and chloroform/methanol soluble proteins (CM-like), such as the α-amylase/trypsin inhibitor family, β-amylase and serpins, were related to wheat allergens, further in vivo analysis will be needed, especially those related to clinical trials in controlled patients, to determine if these lines could be used for food preparation for celiac or other gluten intolerant groups.

Biological significance: Several enteropathies and allergies are related to wheat proteins. Biotechnological techniques such as genetic transformation and RNA interference have allowed the silencing of gliadin genes, providing lines with very low gliadin content in the grains. We report a proteomic-based approach to characterize two low-gliadin transgenic wheat lines obtained by RNAi technology. These lines harbor the same silencing fragment, but driven by two different endosperm specific promoters (γ-gliadin and D-hordein). The comprehensive proteome analysis of these transgenic lines, by combining two-dimensional electrophoresis and RP-HPLC/nESI-MS/MS, provided a large number of spots differentially expressed between the control and the transgenic lines. Hence, the results of this study will facilitate further safety evaluation of these transgenic lines.

Keywords: Celiac disease; Gluten proteins; Low-gliadins; Non gluten proteins; Transgenic wheat; Wheat allergy.