利用双向电泳技术分离节瓜叶片总蛋白的方法研究  

An optimization study on two-dimensional electrophoresis for chieh-qua leaf proteomic analysis

作　　者：赵芹[1] 谢大森[1] 彭庆务[1] 郭巨先[1] 周念[1] 

机构地区：[1]广东省农业科学院蔬菜研究所,广东广州510640

出　　处：《华南农业大学学报》2014年第3期79-85,共7页Journal of South China Agricultural University

基　　金：广东省自然科学基金博士启动基金(10451064001006063);广东省农业科学院院长基金(1279009475432)

摘　　要：【目的】建立适合节瓜叶片蛋白质组学研究的双向电泳体系。【方法】以节瓜品种“A37毛节瓜”为材料，对蛋白质抽提方法、上样量、凝胶浓度及IPG胶条pH范围等关键因素进行探索与优化。【结果和结论】与TCA/丙酮沉淀法及酚提取法相比，改良酚提取法抽提蛋白质总量较高，纯度最高，SDS-PAGE电泳条带明显、清晰，双向电泳图谱蛋白质点最多、清晰均匀、纵横条纹少。利用17 cm pH 4～7范围IPG胶条、120μg蛋白质上样量、12％凝胶浓度，硝酸银染色获得蛋白质点数丰富、分辨率高、背景清晰且重复性好的双向电泳图谱，检测到节瓜叶片蛋白质点1936个，相对分子质量主要分布于15000～100000之间。初步建立了一套适用于节瓜叶片蛋白质组学研究的双向电泳体系，为后续开展蛋白质组学研究及其相关工作奠定了基础。[Objective]To establish two-dimensional electrophoresis （2-DE） system suitable for chieh-qua leaf proteomics analysis .[Method]The effects of various protein extraction methods of 2-DE were inter-compared using the leaves of “A37maojiegua” as the materials, and the loading quantity of sample ,SDS-PAGE gel concentration and the pH range of IPG strips were also investigated and optimized .[Result and conclusion]The results showed that modified phenol extraction method was notably superior to TCA /ace-tone precipitation method and phenol extraction method in total protein quality and clarity of protein bands in SDS-PAGE electrophoresis , and the final 2-DE image map based on modified phenol extraction method had the maxium protein dots and clear background , slight streaking , more completely separated spots compared with the other methods .The further optimization of 2-DE system was carried out to obtain a 2-DE electrophoretogram abundant protein spots , high resolution , clear background and excellent repeat-ability with 17 cm IPG strips,120 μg protein loading quantity , SDS-PAGE with 12% gel concentration and IPG strips with pH 4-7, finally detecting proteins with modified silver nitrate staining .More than 1 936 protein spots were detected and their relative molecular mass ranged from 15 000 to 100 000 .The optimization of the experimental conditions of 2-DE system suitable for chieh-qua proteomics research has been constructed , which provides a basis for further investigation of chieh-qua proteomics researches .

关 键 词：节瓜 叶片总蛋白 蛋白质组学 双向电泳 

分 类 号：S336[农业科学—作物遗传育种]