多子房小麦蛋白质双向电泳体系的建立及优化

为建立适用于显性多子房小麦细胞质效应的蛋白质双向电泳体系,以显性多子房小麦材料DUOII与特异细胞质材料TeZhiI杂交的F1幼穗为材料,采用TCA-丙酮法提取蛋白质,并在IPG胶条长度和pH范围、SDS-PAGE凝胶浓度及蛋白质上样量等方面,对多子房小麦幼穗蛋白质双向电泳体系进行了探究与优化.结果表明,本文采用的蛋白质定量方法准确度高(R²=0.9999),确立了17 cm, pH4～7的IPG胶条, 12% SDS-PAGE分离胶,上样量为900 μg的双向电泳方法体系,获得了最适合本研究蛋白质组分析的双向电泳图谱. 经PDQuest 2DE 8.0.1软件分析,2-DE图谱上可分辨出1.444±14个清晰蛋白质点,且重复性较高(95%), 相关系数为0.960. 建立了一套适用于显性多子房小麦细胞质效应研究的蛋白质双向电泳体系.

Optimization of Two-dimensional Electrophoresis System for the Study of Multi-ovary Character in Wheat

In order to establish a suitable two-dimensional electrophoresis (2-DE) system for the study of the cytoplasm effect on dominant multi-ovary character in wheat, a dominant multi-ovary wheat line DUOII was crossed with a special cytoplasm wheat line TeZhiI and the total protein extracted from the young panicles of the F1 progeny were separated by a 2-DE system which was optimized with the size and pH gradient of IPG strips, SDS-PAGE gel concentration and sample loading quantity. The results showed that it was very accurate that the protein quantitative approach used in this paper (R2=09999) and a 2-DE map with low background and high resolution were obtained using the following optimized procedure: extracting the total protein by TCA acetone precipitation method, loading 900 μg protein sample on 17 cm pH4～7 IPG strip, using 12% gel concentration for SDS-PAGE and staining the gels by Coomassie brilliant blue G-250. A total of 1.444±14 protein spots were detected by the PDQuest 2DE 8.0.1 software, and the match rate of protein spots between two repetitive maps reached up to 95% with a high correlation coefficient of 0.960. An optimized two-dimensional electrophoresis profiles was established, which laid a foundation for the further proteomic research of the cytoplasm effect on dominant multi-ovary character in wheat.