Consoli L, Damerval C
Station de Génétique Végétale, UMR320 INRA/INA-PG/UPS, Ferme du Moulon, Gif-sur-Yvette, France.
Electrophoresis. 2001 Aug;22(14):2983-9. doi: 10.1002/1522-2683(200108)22:14<2983::AID-ELPS2983>3.0.CO;2-#.
A two-dimensional (2-D) electrophoresis procedure was optimized to obtain well-resolved and reproducible patterns of zein polypeptides in maize. All zein isoforms obtained through zein-specific extraction were observed from whole meal extracted with a urea/Triton/2-mercaptoethanol solution. Loading the protein samples at the acidic side of the gradient, using an amino acid solution as catholyte and running for a short time period under high Vh reduced shrinking and instability at the basic side of the isoelectric focusing (IEF) gels. Good quality and reproducible 2-D patterns were thus obtained, allowing automatic spot quantification. A linear relationship between spot intensity and zein isoform amount was established for 20 of 22 zeins detected in a 5.5-8.5 pH range using colloidal Coomassie blue staining in one maize line. The analysis of 45 genetically diverse inbred lines allowed the detection of 59 isoforms belonging to the four classes of zeins, and revealed a large qualitative and quantitative variability of individual isoforms. The classical decrease in zein amount in o2 mutant genotype was observed, and could be quantified for every isoform. The improved technique will be useful to dissect the genetic control of zein expression in maize.
优化了二维（2-D）电泳程序，以获得玉米中醇溶蛋白多肽清晰可辨且可重复的图谱。在用尿素/ Triton / 2-巯基乙醇溶液提取的全粉中观察到了通过醇溶蛋白特异性提取获得的所有醇溶蛋白亚型。将蛋白质样品加载到梯度的酸性一侧，使用氨基酸溶液作为阴极电解液，并在高电压下短时间运行，减少了等电聚焦（IEF）凝胶碱性一侧的收缩和不稳定性。从而获得了高质量且可重复的二维图谱，实现了斑点的自动定量。在一个玉米品系中，使用胶体考马斯亮蓝染色法，在5.5 - 8.5 pH范围内检测到的22种醇溶蛋白中的20种，建立了斑点强度与醇溶蛋白亚型含量之间的线性关系。对45个遗传多样的自交系进行分析，检测到属于四类醇溶蛋白的59种亚型，并揭示了各个亚型在定性和定量上的巨大变异性。观察到o2突变基因型中醇溶蛋白含量的典型下降，并且可以对每种亚型进行定量。改进后的技术将有助于剖析玉米中醇溶蛋白表达的遗传控制。
