萝卜蛋白组分析中叶片蛋白质高效提取方法

Effective Method of Leaf Protein Extraction for Proteomic Analysis in Radish

以萝卜‘NAU-JLQX09’叶片为材料,分别采用Tris-HCl/TCA-丙酮沉淀法和改进的PEG分级沉淀法提取蛋白质,通过双向凝胶电泳技术比较两种方法的效果。结果表明,应用Tris-HCl/TCA-丙酮沉淀法提取的蛋白2-DE图谱可识别出640个蛋白点;而改进的PEG分级沉降法的蛋白组分(F1,F2以及F3)2-DE图谱可清晰识别蛋白点数目分别为537,170与852,总蛋白点数目多且高丰度蛋白RuBisCO主要被沉降在F2中,F3中分离到更多的低丰度蛋白点。因此,在进行萝卜叶片蛋白质组分析时,采用PEG分级沉降法提取叶片蛋白,可获得较高质量的2-DE凝胶图谱。研究结果为应用蛋白质组学解析萝卜重要性状形成分子基础提供了良好的技术支持。

In this study,two protein extraction methods,Tris-HCl/TCA-Acetone and PEG Fractionation,were compared with 2-DE using the leaves of radish line＇NAU-JLQX09＇.It was shown that with the Tris-HCl/TCAAcetone method,the number of protein spots that could be identified by 2-DE profile was 640,while with the modified PEG Fractionation method,the number of protein spots identified in three fractions（F1,F2 and F3） by 2-DE were 537,170 and 852,respectively.Moreover,the major high-abundant proteins like ribulose-1,5-bisphosphate carboxylase/oxygenase（RuBisCO） in leaves were successfully fractioned in F2,indicating that a wide variety of low-abundant proteins could be detected in F3.Therefore,it could be concluded that the 2-DE profile with the PEG Fractionation method was more effective than that with the Tris-HCl/TCA-Acetone method.These results provided an effective proteomic technical support to elucidate the molecular basis of some important agronomic traits using proteomic technique in radish.