| 摘要: |
| 【目的】建立一套适合黄瓜叶片细胞壁蛋白质组学研究的双向电泳体系，为黄瓜细胞壁蛋白质组学的后续研究奠定基础。【方法】以黄瓜品种“长春密刺”幼苗叶片为材料，比较2种不同提取介质（CaCl2和LiCl溶液）对细胞壁蛋白的提取效果，对比IPG胶条梯度、裂解液、上样量及等电聚焦程序对黄瓜叶片细胞壁蛋白双向电泳结果的影响。【结果】以0.2 mol/L CaCl2和3 mol/L LiCl溶液依次提取黄瓜叶片细胞壁蛋白，选用17 cm pH 3～10 NL IPG胶条和裂解液Ⅱ（7 mol/L尿素、2 mol/L硫脲、40 g/L CHAPS、10 g/L DTT和1 mmol/L PMSF），上样量800 μg，按聚焦程序Ⅰ（20 ℃水化14 h，100 V 30 min，250 V 30 min，500 V 30 min，1 000 V 1 h，线性升压5 h至10 000 V，10 000 V聚焦6.5 h）聚焦后进行双向电泳分离和考马斯亮蓝G 250染色，可获得重复性好、分辨率高的双向电泳图谱。【结论】建立并优化了适合于黄瓜叶片细胞壁蛋白质组学分析的双向电泳体系。 |
| 关键词: 黄瓜 叶片 细胞壁蛋白 双向电泳 条件优化 |
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| 基金项目:国家自然科学基金面上项目（31071791）；西北农林科技大学基本科研业务费专项（QN2009011） |
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| Establishment of a two-dimensional electrophoresis system for cell wall proteomics analysis of cucumber leaves |
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| Abstract: |
| 【Objective】This study aimed to establish a two-dimensional electrophoresis（2-DE）system for cell wall proteomic analysis of cucumber leaves.【Method】In the experiment,cucumber seedling leaves of Changchun Mici variety were used as material.Two different extraction buffers for cell wall proteins of cucumber leaves were compared,and factors including pH gradient of IPG strip,different lysis buffers,sample loading levels and isoelectric focusing conditions influencing 2-DE system were examined systematically.【Result】The optimized system included the following steps:(1) extracting the cell wall proteins from cucumber leaves continuously by 0.2 mol/L CaCl2 and 3 mol/L LiCl;(2) separating the proteins with 17 cm pH 3-10 NL IPG strips;(3) using the lysis buffer Ⅱ (7 mol/L urea,2 mol/L thiourea,40 g/L CHAPS,10 g/L DTT and 1 mmol/L PMSF);(4) loading protein samples of 800 μg followed by isoelectric focusing program Ⅰ (20 ℃ hydration 14 h,100 V 30 min,250 V 30 min,500 V 30 min,1 000 V 1 h,5 h gradient 10 000 V and 6.5 h at 10 000 V),and (5) staining the gels by Coomassie Brilliant Blue G250 after SDS-PAGE electrophoresis (125 g/L).Reproducible profiles with high resolution were obtained by the optimized 2-DE system for cell wall proteins of cucumber leaves.【Conclusion】The optimized 2-DE system for cell wall proteins of cucumber leaves was established and it was suitable for proteome comparison analysis. |
| Key words: cucumber leaves cell wall proteins two-dimensional electrophoresis (2-DE) condition optimization |
