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17cm、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17cm、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泉.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 obtain clear two-dimensional electrophoresis (2-DE) image with highest resolution of Rhizoctonia solani, three protein extraction protocols were compared. After analyzing the 2-DE protein images by the software of PDQuest8.0, 1278, 885and827 protein spots were obtained separately by the methods of phosphate solubilization, TCA/acetone precipitation and acetone precipitation, the IPG strip is pH 5-8,17 cm. 2-DE images with clearer background were observed and more protein spots were obtained by phosphate solubilization compared to other protocols, which was due to effective removal of salt and small molecular from the protein sample. Both protocols of TCA/acetone precipitation and acetone precipitation resulted in partial loss of protein spots in the atlas of alkalinity end. In the study, a set of methods were established for reliable protein extraction and 2-DE analysis for Rhizoctonia solani, and laid foundation of further study on the proteomics of Rhizoctonia solani.
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