红麻细胞质雄性不育系与保持系总蛋白提取及双向电泳体系优化

The Total Protein Extraction and Two-dimensional Electrophoresis System Optimization of the Kenaf Cytoplasmic Male Sterility and Maintaining

为探讨红麻细胞质雄性不育形成的分子遗传机理,以红麻细胞质不育系L23A和保持系L23B开花期叶片为材料,对其红麻蛋白质双向电泳体系进行研究,比较TCA/丙酮和酚抽提法提取红麻叶片总蛋白,同时优化上样量和样品制备方法,建立了红麻蛋白质的双向电泳技术体系和凝胶成像染色法,利用PDquest软件分析双向电泳凝胶的相关差异蛋白。结果表明:(1)TCA/丙酮法比酚抽提法更适合于ISO-DALT电泳系统;(2)TCA/丙酮提取的红麻叶片粗蛋白平均产量为31.67 mg/g,是酚抽提法的1.77倍,但裂解效率为160 mg/g,是酚抽提法的36%;(3)每根胶条最佳上样量为300μg;(4)考马斯亮蓝G-250比R-250以及银染色法效果更佳,在17 cm×17 cm的凝胶上平均可获得708个蛋白质点。本研究建立的红麻细胞质雄性不育系与保持系总蛋白提取方法及双向电泳体系,可为揭示红麻雄性不育分子机理奠定基础。

To explore the molecular genetic mechanisms of cytoplasmic male sterility（CMS） in Kenaf,we established the total protein extraction protocol and optimized a two-dimensional electrophoresis system.In this study,we used the kenaf leaves of cytoplasmic male sterile line L23A and its Maintainer L23B in flowering stage as the materials,and compared the protein extraction methods using TCA/acetone and phenol.We also optimized the sample loading amount and sample preparation approach,established a protein two-dimensional electrophoresis system and gels staining method,analyzed the different protein maps with PDQuest image software.The results showed that TCA/acetone method was more suitable for the ISO-DALT electrophoresis system than the phenol extraction method.The average protein productivity extracted by TCA/acetone method was 31.67 mg/g,1.77 times than by phenol extraction method,but the protein disintegrating efficiency was 160 mg/g,only 36% of the phenol extraction method.The best sample loading volume was 300 μg per gel,the staining results of Coomassie Brilliant Blue G-250 was better than that of R-250 and silver staining,and the average number of the protein spots was 708 in per gel.Therefore,the two-dimensional electrophoresis system that we established could provide a basis for exploring the molecular genetic mechanisms of CMS in Kenaf.