梨叶片总蛋白提取及双向电泳体系的建立  

Total protein extraction and establishment of two-dimensional electrophoresis system of pear leaf

作　　者：胡敏[1] 徐凌飞[1] 张军科[1] 仇宗浩[1] 丁勤[1] 张静[1] 

机构地区：[1]西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2012年第1期197-203,209,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31171925);国家梨产业技术体系建设专项(CARS-29)

摘　　要：【目的】探索建立有效的梨叶片蛋白质组双向电泳体系,为梨蛋白质组的后续研究提供参考。【方法】以早酥梨叶片为材料,比较蛋白质提取方法、蛋白质裂解时间、IPG胶条的pH梯度和长度、等电聚焦程序及SDS-PAGE电泳参数对梨叶片蛋白双向电泳结果的影响。【结果】采用第2种改良的三氯乙酸(TCA)/丙酮沉淀法提取梨叶片总蛋白,蛋白质裂解1h,选用长17cm、pH 4～7的IPG胶条及聚焦程序Ⅱ进行等电聚焦,于100V30min、180V 6h条件下进行SDS-PAGE垂直电泳,可以获得背景清晰、重复性好的双向电泳图谱。【结论】建立并优化了适用于梨叶片蛋白质组分析的双向电泳体系。【Objective】This study was to develop an efficient two-dimensional electrophoresis system of protein for pear leaf.【Method】In this experiment,the leaf of ‘Zaosu’ pear（Pyrus bretschneideri Rehd.） was chosen as material.The factors including the extraction methods,time of dissolving proteins,types of IPG strip,focusing procedure,and SDS-PAGE electrophoresis procedure that influence two-dimensional electrophoresis（2-DE）system were examined systematically.【Result】The optimized system used the following procedure：extracting the total protein from pear leaf by method 2 precipitation,dissolving proteins time being 1 h,separating the proteins with 17 cm pH 4-7 IPG strips,isoelectric focusing with IEF 17 cm programⅡ,and SDS-PAGE program（100 V 30 min,180 V 6 h）.Reproducible 2-DE maps with high resolution,clear background intensity and slight streaks were obtained by this optimized 2-DE system of pear leaf.【Conclusion】The optimized two-dimensional electrophoresis system of protein from pear leaf was established,which was suitable for proteome comparison analysis.

关 键 词：梨 叶片 蛋白质提取 双向电泳 

分 类 号：S661.2[农业科学—果树学]