| 适用于蛋白质双向电泳的棉花胚性培养物蛋白质提取技术 |
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| 引用本文： | 曹景林,朱龙付,谭家福,邓锋林,李允静,郝娟,徐士成,张献龙.适用于蛋白质双向电泳的棉花胚性培养物蛋白质提取技术[J].棉花学报,2009,21(1):3-9. |
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| 作者姓名： | 曹景林 朱龙付 谭家福 邓锋林 李允静 郝娟 徐士成 张献龙 |
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| 作者单位： | 华中农业大学作物遗传改良国家重点实验室,武汉,430070 |
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| 基金项目： | 教育部跨世纪优秀人才培养计划 |
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| 摘 要： | 以陆地棉品种Coker 201为材料，对与甲醇/醋酸铵丙酮沉淀结合的酚抽提法和裂解液配方进行了优化，建立了适用于蛋白质双向电泳体系的棉花胚性培养物蛋白质样品制备技术。对胚性培养物，通过添加PVPP研磨和丙酮、TCA/丙酮、甲醇/醋酸铵等有机溶剂的系列洗涤，然后再进行酚抽提，蛋白质质量得到显著改善，获得的2-DE图谱质量高、可分辨的蛋白质点数多。比较分析3种裂解液对蛋白质的溶解效果发现，在裂解液中混合采用7 mol·L-1尿素、2 mol·L-1硫脲以及4% CHAPS是最有效的配方。
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| 关 键 词： | 棉花 胚性培养物 蛋白质 双向电泳 |
Protein Extraction from Embryogenic Cultures of Cotton for Two-dimensional Gel Electrophoresis |
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| CAO Jing-lin,ZHU Long-fu,TAN Jia-fu,DENG Feng-lin,LI Yun-jing,HAO Juan,XU Shi-cheng,ZHANG Xian-long.Protein Extraction from Embryogenic Cultures of Cotton for Two-dimensional Gel Electrophoresis[J].Cotton Science,2009,21(1):3-9. |
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| Authors: | CAO Jing-lin ZHU Long-fu TAN Jia-fu DENG Feng-lin LI Yun-jing HAO Juan XU Shi-cheng ZHANG Xian-long |
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| Institution: | National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan,Hubei 430070, China |
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| Abstract: | A new preparation protocol of protein extracts was established for 2-DE of embryogenic culture proteins of Gossypium hirsutum,cv. Coker 201, by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation and optimized lysis buffer. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning and TCA/acetone washes followed by aqueous methanol/ammonium acetate and acetone lavation before phenol extraction. The protocol gave a higher protein yield and higher resolution and spot intensity in 2-DE analysis. Furthermore, three lysis buffers for 2-DE were compared on cotton embryogenic calli proteins isolation. Incorporation of chaotrope mixture (7 mol·L-1 urea, 2 mol·L-1 thiourea) with 4% CHAPS was found to be the most effective step. |
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| Keywords: | embryogenic cultures protein two-dimensional gel electrophoresis |
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