不同提取液提取水稻幼苗质外体蛋白效果的比较

提取植物组织质外体蛋白质的主要困难是提取效率低且易被细胞质蛋白污染。为解决上述问题,以12天93-11水稻幼苗为试验材料,使用3种含不同浓度钾和钙离子的缓冲液作为提取液进行提取效果比较。3种提取液的相同成分都是0.1mol/L Tris-HCl pH 7.6,1mmol/L PMSF,区别点在于:Buffer A含0.2mol/L KCl;Buffer B含0.2mol/L CaCl₂;Buffer C含0.1mol/L KCl和0.1mol/L CaCl₂。结果表明,Buffer A的蛋白产率达到了(0.49±0.07)mg/g FW(叶片)和(0.83±0.06)mg/g FW(根部),比Buffer B和Buffer C分别提高了122.7%和53.1%(叶片)以及102.4%和59.6% (根部)。六磷酸葡萄糖脱氢酶活性检测的结果表明在这些蛋白质提取物中细胞质蛋白的污染率很低,可以控制在1%以下。这些实验结果说明通过优化提取液,建立了有效提取水稻幼苗质外体蛋白质的方法,可应用于植物质外体蛋白质组学研究。

Comparison of the Extraction Effects of Rice Seedling Apoplast Proteins Using Different Extraction Buffers

To date, low extraction efficiency and cytoplasmic contamination is a great problem that apoplast protein extraction faces. To effectively extract apoplast proteins, using 12 day-old 93-11 rice seedlings, the extraction efficiency and cytoplasmic contamination ratio of the apoplast protein extracts by three different extraction buffers (Buffer A, Buffer B and Buffer C) were compared. The three buffers all contain 0.1mol/L Tris-HCl pH 7.6 and 1mmol/L PMSF, however, Buffer A also contains 0.2mol/L KCl ; Buffer B also contains 0.2mol/L CaCl₂; and Buffer C also contains 0.1mol/L KCl and 0.1mol/L CaCl₂. The results showed that the protein yield of buffer A reached (0.49±0.07)mg/g FW(leaf)and (0.83±0.06)mg/g FW (root), respectively, 122.7% (leaf) and 102.4% (root) greater than Buffer B, and 53.1% (leaf) and 59.6% (root) greater than Buffer C. The glucose-6-phosphate dehydrogenase (G6PDH) enzyme assay showed that the cytoplasmic contamination ratios of these apoplast protein extracts were very low, below 1%, which could be considered as negligible contamination. Therefore, by optimizing the extraction buffer, an effective extraction method for apoplast proteins was established, which could be applied to the plant apoplast proteomic studies.