水稻叶片质膜的纯化及质膜蛋白质双向电泳分析
Purification of Plasma Membrane from Rice Leaves and Analysis of the Proteins by Two-Dimensional Electrophoresis
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摘要: 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Dextran T 500/ PEG 3350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VO 43--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Brij 58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-D SDS-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。Abstract: An effective method for the isolation of plasma membrane (PM) from rice,Oryza sativa,seedlings was established using aqueous two-phase system which was composed of Dextran T 500 and PEG 3350. The effect of the concentration of the polymer on partition of PM in polymer system was studied．Phase separation was carried out in a series of two-phase systems containing 6.1%-6.5% (w) of each polymer dissolved in phase buffer. The result indicated that the most effective phase partition system which consisted of 6.3%/6.3%(w) PEG 3350/Dextran T 500 in 0.25 mol/L sucrose, 5 mmol/L potassium phosphate and 3 mmol/L KCl (pH 7.8) was suitable for the isolation of PM from rice. The electron micrograph stained with uranyl actate-lead citrate stain and marker enzyme activities analysis proved that the isolated PM had obtained high purity (93.5%) and right-side out sealed vesicles. Purified PM proteins were also obtained from crude PM in good yield (2.73%). To improve the solubilization of hydrophobic PM proteins and dissociate protein complexes, PM proteins were treated by an optimized rehydration buffer including new zwitteronic detergents, just as ASB-14 and CHAPS. 1-D SDS-PAGE of purified PM proteins showed more band numbers and well-proportioned bands intensity than that of crude PM. PM proteins were also separated by IEF/SDS-PAGE. 579±17 well-resolved proteins with a much clearer background were obtained, which also showed good reproducibility among three independent experiments. The result above showed that the method to purify PM proteins using the aqueous two-phase partition system could be suitable for high-throughput PM proteomic analysis, such as SDS-PAGE combined with HPLC-ESI-MS/MS and 2-DE analysis.
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