玉米籽粒蛋白质双向电泳技术体系的优化

摘要

为了获得玉米籽粒双向电泳中蛋白质提取的最佳方法，建立最佳的玉米籽粒蛋白双向电泳技术体系，对酚抽提法、三氯乙酸／丙酮沉淀法（ TCA／丙酮沉淀法）和可溶性蛋白提取法3种不同提取方法得到的蛋白质进行了分析，并在相同条件下进行双向电泳比对分析，结果表明：可溶性蛋白提取法获得的蛋白纯度高、浓度适中、双向电泳蛋白质点最丰富，最适合双向电泳研究；利用可溶性蛋白提取法获得的蛋白，对第一向等电聚焦参数等条件，以及不同pH值范围的IPG预制凝胶条进行了比较分析，结果表明：采用pH值3～10的IPG预制凝胶条时，采取电压梯度上升的方式，总聚焦70000 Vhs、上样量800μg效果最佳，采用pH值4～7的IPG预制凝胶条时，总聚焦80000 Vhs、上样量900μg效果最佳，并且采用pH值4～7的IPG预制凝胶条相比pH值3～10的凝胶条能分离到更多的点，最后结合蛋白质点MALDI-TOF-TOF质谱鉴定分析，建立了一套适用于玉米籽粒的蛋白质双向电泳技术方法，即采用可溶性蛋白提取法提取蛋白，采用24 cm、pH值4～7的IPG干胶条，上样900μg电泳效果最佳，等点聚焦程序：500 V、1 h，1000V、1 h，2000 V、1 h，4000 V、2 h，8000 V、4 h，8000 V、80000 Vhs，500 V保持。试验研究为后续玉米籽粒发育差异蛋白研究奠定了技术基础。%In order to obtain the best protein extraction methods of maize grain for 2-DE,and establish the best grain protein 2-DE technology system ,quality of the proteins extracted by 3 different extraction methods were ana-lyzed,and analysis of 2-DE for comparison in the same condition was done ,results showed that the protein extracted by soluble protein extraction method had high purity ,moderate concentration and the most abundant protein spots of 2-DE,was most suitable for the study of 2-DE;in order to determine the most appropriate isoelectric focusing condi-tions,isolated distribution clearer protein spots,use the protein extracted by soluble protein extraction method,pro-tein electrophoresis sample quantity and IEF parameters were optimized ,the most suitable pH range of IPG precast gel for maize grain protein was determined ,binding protein MALDI-TOF-TOF mass spectrometry analysis,a set of protein 2-DE technique method suitable for maize grain was established,that is,protein extraction use the soluble protein extraction method,isolated distribution use the 24 cm,pH 4-7 IPG dry strip,the proper amount of sample for electrophoresis was 900 μg,IEF procedures:500 V,1 h,1 000 V,1 h,2 000 V,1 h,4 000 V,2 h,8 000 V, 4 h,8 000 V,80 000 Vhs,500 V keep.It was laid the foundation for the further study of grain development related proteins.