利用双向电泳技术分离大豆矮秆突变体相关蛋白

Isolation of Proteins Related to the Dwarf Mutant by Two-dimensional Gel Electrophoresis in Soybean

矮秆是大豆育种的重要目标性状之一。以大豆野生型东农42和矮秆突变体东泽11为材料,利用近年来发展起来的双向电泳技术,在蛋白质水平对两个材料的差异蛋白质进行筛选,目的是鉴定与矮秆突变体相关的蛋白,为基因克隆提供依据。通过对酚(Phenol)法与TCA/丙酮沉淀法二种提取方法、100μg和200μg两种加样量、考马斯亮蓝染色和银染两种染色方法的比较,发现用丙酮沉淀法提取叶片可溶性总蛋白、加样量为200μg进行电泳,用考马斯亮蓝染色的效果较好,从而建立了大豆叶片总蛋白双向电泳技术优化体系。用该体系对野生型与突变体叶片全蛋白的差异分析,鉴定出9个蛋白差异点,其中6个上调表达,3个下调表达。

Dwarf is one of the major target traits in soybean breeding. Two dimensional electrophoresis （2- DE） was used to analyze the different proteins between the dwarf mutant （Dongzell） and its wild type （Dongnong42）, which can be used to identified the gene related to the dwarf mutant. By combination and optimization of various conditions, a sample preparation method of 2-DE technique was established, which were used for further analyses. The total leaf proteins between mutant and wild soybean were compared with two- dimensional gel electrophoresis. There were 9 spots were notable detected, including 6 up-regulated proteins and 3 down-regulated proteins. The two dimensional electrophoresis （2-DE ） system to analyze leaves was established. The similarity and difference of this system from the previous were discussed.