| 摘要: |
| 【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Rubisco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。 |
| 关键词: 蛋白质组 小麦叶片 分离技术 Western blot 双向电泳 |
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| 基金项目:公益性行业（农业）科研专项(200903035-02)；高等学校学科创新引智计划项目(B07049) |
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| Study of a rapid method for depletion of highly abundant protein Rubisco enzyme from wheat (T.aestivum) leaf for proteome analysis |
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| Abstract: |
| 【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. |
| Key words: proteome wheat leaf fractionation technique Western blot Two-Dimension electrophoresis (2-DE) |
