圆果种黄麻功能叶总蛋白提取方法及双向电泳体系的优化

摘要

蛋白质提取方法和电泳条件是蛋白质组学研究中双向电泳的关键.本文初步建立起一套适合圆果种黄麻功能叶总蛋白双向电泳(2-DE)分析的高效提取方法及电泳条件,在双向电泳技术中,比较了TCA丙酮、Tris-base/丙酮、Tris-HCI的蛋白提取方法,并优化了上样量和样品制备方法.结果表明,TCA丙酮法提取蛋白质产量高,达79.0 mg91,比Tris-base/丙酮法和Tris-HCl的产量分别高出44.2%和114.1%.该法得到的2-DE图谱背景清晰,蛋白点数最多,约602个,较其他两种方法更适合用于黄麻功能叶总蛋白双向电泳分析;350ug为最佳上样量.这为黄麻蛋白质组学后续研究奠定了基础.%The protein extraction methods and electrophoresis conditions are the key facts of two-dimensional electrophoresis in proteomics.In this study, in order to set up a suitable method for jute leaf total protein extraction and the optimized condition for two-dimensional electrophoresis, we compared the protein extraction methods using TCA acetone, Tris-base acetone and Tris-HCl.We also optimized the sample loading amount and sample preparation approach.The results showed that protein productivity reached 79.0 mg g–1 at most by TCA acetone method, and 44.2％ and 114.1％ higher than by other two methods, respectively.Besides, the 2-DE image background was the clearest and the protein spots were the most reaching about 602 by TCA acetone method, which is much more suitable as extraction method for jute leaf protein two-dimensional electrophoresis.The most suitable sample loading quantity was 350 μg, which lays a foundation for further study of jute proteomics.The construction of two-dimensional electrophoresis system can be use to study the differences of protein expression on normal material and mutant of jute, and promote the application of proteomics in jute mutant.