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水稻叶片质膜的纯化及质膜蛋白质双向电泳分析

DOI: 10.7671/j.issn.1001-411X.2012.01.003, PP. 11-17

Keywords: 水稻,质膜,双水相分配法,双向电泳

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Abstract:

采用双水相分配法和离心法对水稻叶片的细胞质膜进行了纯化.选用聚合物DextranT500/PEG3350质量分数分别为6.1%、6.2%、6.3%、6.4%、6.5%的双水相体系,对3次分配后的细胞质膜纯化效果进行了研究,并进行了细胞质膜标志酶H+-ATPase的活性测定、柠檬酸铅-醋酸铀双染色及电镜检测。结果表明:聚合物质量分数为6.3%的双水相体系可以获得高纯度的质膜微囊,且随分配次数的增加,可以有效减少其他内膜的污染,其质膜标志酶VO43--ATPase的相对活性高达93.5%。粗质膜经过3次两相分配后的蛋白质产率为2.73%。非离子型去垢剂Brij58对质膜H+-ATPase的活性变化影响结果表明,纯化的质膜微囊封闭性较好,主要为正向型质膜微囊。质膜蛋白质经增溶缓冲液溶解及1-DSDS-PAGE的结果表明,纯化后的质膜有效减少了特定蛋白质条带的丰度,使蛋白质条带的分布更为均匀。双向电泳结果表明,纯化后的质膜双向电脉图谱具有低背景、高分辨率和重复性的优点,为进一步的水稻质膜蛋白质组研究提供了可靠的试验材料。

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