蚜虫蛋白质组双向电泳体系条件优化

Optimization of two-dimensional gel electrophoresis technology system for aphid proteome

为优化有效分离蚜虫全蛋白的双向电泳体系,以豌豆蚜Acyrthosiphon pisum(Harris)为材料,比较了不同蛋白提取方法(裂解液提取法、三氯乙酸/丙酮沉淀法、饱和酚抽提法)、蛋白上样量、IPG胶条p H梯度等条件对蚜虫全蛋白双向电泳的影响。结果表明:不同的蛋白提取方法所获得的豌豆蚜全蛋白提取率有显著差异,其中以饱和酚抽提法的提取率最高,为5.34±0.26 mg/g,且所提取的蛋白种类丰富度也优于其它2种方法;当蛋白上样量为100μg时所得蛋白点为1 793±36个,高于其它上样量;相同条件下选用11 cm p H 3~10非线性IPG胶条时所得蛋白点为1 803±42个,优于p H 4~7的IPG胶条。表明采用饱和酚抽提法进行提取蛋白,选用11 cm p H 3~10非线性IPG胶条,以蛋白银染方法、分离胶浓度为12.5%进行双向电泳时最适蛋白上样量为100μg,所得可识别蛋白质点最高,且重复性试验匹配率高,可用于豌豆蚜蛋白质组学研究。

This study aims to optimize a two-dimensional electrophoresis( 2-DE) system for studying total protein of aphids. In the experiment,the pea aphid Acyrthosiphon pisum( Harris) was used. Three different methods of protein extraction,namely lysate extraction,TCA-acetone precipitation and saturated phenol extraction,were compared. The factors including loading amount of protein samples and two kinds of different IPG strips( p H 3- 10 nonlinear and p H 4- 7) were tested. The results showed that there were significant differences among the different extraction methods. The saturated phenol extraction yielded the highest amount of 5. 34 ± 0. 26 mg / g protein as well as the best richness of proteins. For aphid 2-DE proteome research,the following protocol should be adopted: p H 3- 10 nonlinear IPG strip,100 μg protein loading amount,12. 5% gel for SDS-PAGE,and silver staining. This method had detected a total of 1 803 ± 42 protein spots with a highly reproducible result in the proteomic study of the pea aphid.