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为建立水稻纹枯病菌菌体蛋白的双向电泳技术,获得分辨度高、重复性好的双向电泳图谱。采用了酚抽提法、TCA/丙酮沉淀法和丙酮沉淀法进行水稻纹枯病菌菌体蛋白质的提取,利用17 cm、pH 5～8的IPG胶条对样品进行双向电泳,用PDQuest 8.0软件对电泳图谱进行分析,确定电泳的最佳参数。结果显示:采用17 cm、pH 5～8的IPG胶条能得到较好的双向电泳图谱;同一样品的三种不同蛋白提取方法(酚抽提法、TCA/丙酮沉淀法和丙酮沉淀法)分别得到1278、885、827个蛋白质点。酚抽提法能有效去除样品中的盐分和小分子的干扰,得到背景清晰的蛋白图谱;TCA/丙酮沉淀法和丙酮沉淀法所得图谱有一定的杂质干扰,同时TCA/丙酮沉淀法和丙酮沉淀法有蛋白点缺失,尤其在碱性端存在明显缺失现象。研究结果建立了一套适于水稻纹枯病菌菌体蛋白的可靠提取方法和双向电泳体系,为进一步研究其蛋白质组学奠定了基础。
To establish two-dimensional gel electrophoresis of Rhizoctonia solani, two-dimensional gel electrophoresis with high resolution and good reproducibility was obtained. Phenol extraction method, TCA / acetone precipitation method and acetone precipitation method were used to extract Rhizopus oryzae bacterial cell proteins. Two-dimensional electrophoresis was carried out using IPG strips of 17 cm and pH 5-8. PDQuest 8.0 software Electrophoresis patterns were analyzed to determine the best parameters for electrophoresis. The results showed that the best two-dimensional gel electrophoresis patterns were obtained with 17 cm and IP 5 ~ 8 pHG strips. Three different protein extraction methods (phenol extraction, TCA / acetone precipitation and acetone precipitation) 1278,885,827 protein spots were obtained. Phenol extraction method can effectively remove the interference of salt and small molecules in the sample to obtain a clear protein profile; TCA / acetone precipitation and acetone precipitation obtained by the map have some impurities, while TCA / acetone precipitation and acetone precipitation Law point of protein loss, especially in the alkaline end there is a clear absence of the phenomenon. The results of the study established a set of reliable extraction methods and two-dimensional electrophoresis system suitable for Rhizoctonia solani to lay a foundation for further study of proteomics.