中文摘要：面筋蛋白是小麦面包烘烤品质的主要决定因素，也是与小麦疾病（包括腹腔疾病（CD）和过敏）相关的重要因素。本文利用两种转基因低醇小麦和非转基因小麦的谷物总蛋白进行了蛋白质组学研究，并评估了基因沉默对靶标蛋白和非靶标蛋白的影响。由于蛋白具有很大的复杂性，因而本文用基于二维电泳凝胶的RP-HPLC/nESI-MS/MS方法对四种核心蛋白组分进行了分离分析：醇溶蛋白亚基、麦谷蛋白亚基、代谢蛋白和类CM蛋白。研究发现，转基因小麦积累的醇溶蛋白、HMW-GS、代谢蛋白和甲醇/氯仿可溶性蛋白高度下调。基于这些数据，结合代谢蛋白和甲醇/氯仿可溶性蛋白与小麦过敏原相关，因而进一步的体内分析十分必要。本文对两种低醇蛋白转基因小麦的蛋白组学进行了研究，发现两个品种有同样的基因沉默片段，但分别有不同的的胚乳特异启动子（γ-醇溶蛋白和d-大麦醇溶蛋白）驱动。基于二维电泳凝胶和高效液相色谱/串联质谱的转基因小麦综合蛋白质组分析，为转基因小麦与非转基因小麦提供了大量差异表达的位点。因此，本研究的结果将有助于这些转基因株系的进一步安全性评价。
外文摘要：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 alpha-amylase/trypsin inhibitor family, beta-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 (gamma-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.
外文关键词：Celiac disease; Gluten proteins; Low-gliadins; Non gluten proteins; Transgenic wheat; Wheat allergy
作者：Dolores Garcia-Molina, Maria; Muccilli, Vera; Saletti, Rosaria; 等.
作者单位：CSIC
期刊名称：JOURNAL OF PROTEOMICS
期刊影响因子：3.914
出版年份：2017
出版刊次：8
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