番茄子叶总蛋白双向电泳体系的建立  

Establishment of Two-dimensional Electrophoresis System of Tomato Cotyledons

作　　者：黎飞[1] 徐秋芳[1] 臧宪朋[1] 赖亿玉[1] 程维舜[1] 徐幼平[2] 蔡新忠[1] 

机构地区：[1]浙江大学农业与生物技术学院生物技术研究所,杭州3100291 [2]浙江大学分析测试中心,杭州310029

出　　处：《园艺学报》2010年第4期661-668,共8页Acta Horticulturae Sinica

基　　金：‘973’计划项目(2009CB119000);国家自然科学基金项目(30671352;30200179);浙江省自然科学基金杰出青年团队项目(R307396);教育部霍英东教育基金会高等院校青年教师基金项目(101032)

摘　　要：通过对IPG胶条梯度、上样方式、上样量、聚焦条件等4方面条件的优化,建立了番茄子叶总蛋白双向电泳体系,即采用TCA丙酮沉淀法提取番茄子叶总蛋白,选用24cmpH3-7NL的IPG胶条,采用水化上样,上样量300μg,按聚焦程序Ⅰ或Ⅱ聚焦后进行双向电泳分离和银染染色。若采用制备胶,以获取高含量蛋白点,则将上样量提高至1.5mg,按聚焦程序Ⅲ进行聚焦后进行双向分离,并采用胶体考马斯亮蓝染色。采用该优化的体系可以获得分辨率高、重复性好的双向电泳图谱。A two-dimensional electrophoresis（2-DE）system for proteomic analysis of tomato cotyledons was established by optimizing the parameters including the pH gradient of IPG strip,sample application,sample loading and isoelectric focusing conditions. The optimized system include the following steps：Extracting the total protein from tomato cotyledons by TCA/acetone precipitation, separating the proteins with 24 cm pH3-7NL IPG strips,loading protein samples of 300 μg by rehydration method followed by isoelectric focusing programⅠorⅡ,and staining the gels by silver staining after SDS-PAGE electrophoresis. When preparative gel was applied,in order to obtain abundant protein spots, 1.5 mg sample was loaded followed by the isoelectric focusing program Ⅲ,and staining with colloidal Coomassie Brilliant Blue. Reproducible profiles with high resolution were obtained by this optimized 2-DE system of tomato cotyledon.

关 键 词：番茄 双向电泳 条件优化 子叶 蛋白质 

分 类 号：S641.2[农业科学—蔬菜学]