黄瓜叶片细胞壁蛋白质提取方法的筛选

Selection for the Extraction Metheds of Cucumber Leaf Cell Wall Proteins

以黄瓜幼苗的叶片为材料,分别采用酚提取法、盐提法、尿素提取法和真空渗透提取法分离细胞壁蛋白,比较不同提取方法的提取效果,以期为黄瓜细胞壁蛋白质组学研究的开展奠定基础。结果表明:盐提法的蛋白产率最高,尿素提取法次之,真空渗透提取法和酚提取法的蛋白产率较低;采用六磷酸葡萄糖脱氢酶活性测定法检测细胞质蛋白的污染率,其中,真空渗透提取法获得的细胞壁蛋白污染率最低(1.07%),其次为盐提法(2.17%),而酚提法和尿素提取法的污染率分别高达25.90%和2.60%;SDSPAGE电泳分析表明盐提法的蛋白条带清晰,且细胞质中高丰度蛋白Rubisco的条带不明显。

The extraction effects of the cell wall protein in cucumber seedling leaves with 4 different methods, phenol extraction method, salt extraction method, urea extraction method and vacuum infiltration extraction method, were compared to establish a foundation for the subsequent cucumber cell wall proteomics study. The results showed that protein yield extracted by salt extraction method was the highest, followed by urea extraction method, and vacuum infiltration and phenol extraction method were the lowest. The cytoplasmic protein contamination rate was detected by glucose-6-phosphate dehydrogenase activity assay. The vacuum infiltration extraction method obtained the lowest contamination rate of cell wall protein(1.07%), followed by salt extraction method(2.17%), while the contamination rate of phenol and urea extraction method was up to 25.90% and 2.60%,respectively. SDS-PAGE gel analysis showed that salt extraction method provided clear protein bands and no obviously high abundance cytoplasm protein Rubisco bands were found.