利用双向电泳技术分离野牛草叶片蛋白的方法研究  

Key Technology of Separation of Buffalograss Leaf Proteome by Applying Two-dimensional Gel Electrophoresis

作　　者：康俊梅[1] 熊军波[1] 孙彦[2] 李燕[1] 沈静[3] 杨青川[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]中国农业大学草地研究所,北京100193 [3]甘肃农业大学,兰州730070

出　　处：《西北农业学报》2010年第2期29-34,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家科技支撑计划(2006BAD01A19-3);科研院所基本科研业务专项经费(2010jc-13)

摘　　要：制备野牛草叶片蛋白质样品,对双向电泳条件等技术环节进行优化,以期获得高分辨率、重复性好的蛋白质双向电泳图谱,建立适应野牛草叶片蛋白组学研究的技术平台,并对其蛋白组进行初步分析。采用TCA/acetone沉淀法和Trizol沉淀法分别提取野牛草叶片蛋白,通过不同聚焦时间分离,最终经硝酸银染色,以Imagemaster 2D Platinum V 5.0图像分析软件对所得的双向电泳图谱进行分析比较。对获得的最佳图谱进行蛋白点分布分析,选择最适合的胶条pH值。结果表明：Trizol沉淀法提取蛋白样品,总聚焦伏时数在8-10万V.h间,为最适合条件,获得图谱背景清晰,纵横纹少,蛋白点分离较完全,在pH3-10胶条凝胶可检测到的蛋白质点达到800个。对蛋白点pH分布范围分析表明,85%蛋白点分布在pH4-7之间,选择pH4-7的IPG胶条,能检测到1 000个以上的蛋白点。以Trizol沉淀法结合优化的双向电泳参数对野牛草叶蛋白组进行双向电泳分离,获得了高分辨率、高重复性的双向凝胶电泳图谱,可用于野牛草差异蛋白质组学研究。The study based on the optimized of buffalograss leaf proteome preparation and the 2-DE protocol gets a high resolution and reproducibility 2-DE image,which will be beneficial for a further study the buffalograss proteomics.The proteins of buffalograss leaf were extracted by trichloroacetic acid /acetone（TCA/A） precipitation and Trizol-base method.The proteins were separated by different 2-DE isoelectric focusing protocol,stained by Silver-staining and the finally 2-DE image analyzed and compared using Image Master 2D Platinum Version 5.0 Analysis Software.Results showed that Trizol-based method for sample preparation,and with a total isoelectric focusing at 80000vhr,the final 2-DE map with a clear background,little streaking,more completely separated spots and there were more 800 spots were detected on the 2-DE gel with pH 3～10 nonlinear gradient strip and 10% SDS-PAGE gel using Silver-staining.Spot grouping according to the pI show that 85% located at pI 4～7.Taking a pH 4～7 linear range strips as a changed,there were more then 1000 spot detected.Trizol-base method combining with the optimization isoelectric focusing protocol,we get a high resolution and more reproducibility 2-DE image,more then 1000 spots were detected on the 2-DE gel,and it suggest that the optimized method and protocol is expected to have excellent applications in proteomics studies of buffalograss.

关 键 词：野牛草 蛋白质组 双向电泳 

分 类 号：S543.9[农业科学—作物学]