小麦叶片蛋白质组分析中高丰度蛋白Rubisco酶的快速去除方法研究

Study of a rapid method for depletion of highly abundant protein Rubisco enzyme from wheat(T.aestivum) leaf for proteome analysis

【目的】建立一种小麦叶片蛋白质组分析时去除全蛋白提取物中高丰度蛋白Rubisco酶的方法,为富集低丰度蛋白,提高双向电泳灵敏度提供技术支持。【方法】采用分离技术,以铭贤169小麦苗期叶片为材料,使用不同浓度(1,2,5,10 mmol/L)的肌醇六磷酸钠,结合二水氯化钙在不同温度(4,20,37℃)去除小麦叶片蛋白中的Rubi-sco酶,筛选去除小麦叶片蛋白Rubisco酶的最佳条件,并采用一维凝胶电泳(1-DE)、Western blot方法检测去除Rubisco酶的有效性,用二维凝胶电泳(2-DE)评估该方法对样品在二维凝胶上分辨率的影响。【结果】在37℃条件下,采用10 mmol/L肌醇六磷酸钠结合10 mmol/L的二水氯化钙去除小麦叶片蛋白中的Rubisco酶,可以获得较好的去除效果。在上述条件下作用10 min,小麦叶片水溶性蛋白提取物中超过80%的Rubisco酶得以沉淀去除,二维电泳图谱上有更多的低丰度蛋白点显现,且水平条纹明显减少,分辨率有所提高。【结论】得到了一种用于小麦叶片蛋白质组分析时去除高丰度蛋白Rubisco酶的方法,该方法具有快速、高效、成本低的特点。

【Objective】 This study established a rapid method for removing the extremely abundant Rubisco enzyme from wheat leaf soluble protein extract,to provide technical support for increasing the visualization of less abundant proteins on 2-DE gels,and subsequently to improve the resolution of 2-DE gel.【Method】 Rubisco removal was achieved through fractionation techniques,using different concentrations of Ca2＋ and phytate（0,1,2,5,10 mmol/L） to precipitate Rubisco from wheat leaf soluble protein extract at different temperatures（4,20,37 ℃）,thus,we screened the optimum condition of rubisco removal from wheat leaf soluble proteins.One-Dimension electrophoresis（1-DE）,Western blot and Two-Dimension electrophoresis（2-DE） were employed to assess the efficiency of this method,respectively.【Result】 The result showsed 10 mmol/L Ca2＋ and 10 mmol/L phytate can effectively precipitate Rubisco at 37 ℃,after incubation for 10 min,more than 80% of the extremely abundant Rubisco enzyme from wheat leaf soluble protein extract can be removed.This subsequently increased the visualization of less abundant proteins and reduced horizontal streaking,and increased the resolution of 2-DE gel.【Conclusion】 We got a method to remove the extremely abundant Rubisco enzyme from wheat leaf soluble protein extract for proteomics analysis,and this method is not only rapid and valid,but also inexpensive.