摘要
以芥菜型油菜黄化突变体L638-y及其野生型L638-g五叶期叶片为材料,用IEF/SDS-PAGE双向凝胶电泳技术对两种不同蛋白质提取方法(TCA/丙酮沉淀法和改进的PEG分级沉淀法)进行了比较,同时在IPG胶条pH范围及SDS-PAGE胶浓度选择上进行了探索与优化.结果表明,以pH4～717cm的线性IPG胶条进行IEF,11%SDS-PAGE进行第二向电泳,每350μl体系上样量为180μg,蛋白质可以得到较好的分离,2-DE图谱质量最佳.用改进的PEG分级沉淀法提取的突变体L638-y叶片总蛋白的2-DE图谱可清晰识别的蛋白质点数目为(1235±6)个,比TCA/丙酮沉淀法多识别出330个蛋白质点;用该方法提取蛋白质时,在突变体L638-y与其野生型L638-g叶片总蛋白2-DE图谱上可识别出差异蛋白质点数目为190个,比用TCA/丙酮沉淀法提取蛋白质时多鉴别出100个差异蛋白质点.由此表明,研究芥菜型油菜黄化突变体L638-y叶片蛋白质组变化,采用改进的PEG分级沉淀法提取蛋白质更为简单有效.
Proteomics has become a powerful technology being successfully used in plant science research to investigate different biological processes, including plant genetics, development, physiological ecology and responses to climate change. Two-dimensional electrophoresis （2-DE） is a potential analytical tool in proteomics to identify qualitative and quantitative changes of proteins and reveal the changes of protein expression under different growth conditions. However, protein sample preparation is the key fundamental steps of 2-DE analysis, and it is also a prerequisite for proteomics analysis. L638-y is a chlorophyll-deficient mutant naturally generated from the wild type L638-g in Brassica juncea L., which is typical characterized with yellow leaves and no other different traits from the wild type except leaves color. In order to efficiently analyze the differential expression proteins between the mutant L638-y and its wild type L638-g, two protein extraction methods for proteomic analysis, TCA/acetone extraction method and an improved polyethylene glycol （ PEG） fractionation method, were compared. Total proteins were extracted from leaves of the mutant L638-y and its wild type L638-g at five leaves period with the two protocols, then the samples were analyzed by 2-DE, pH of IPG strip and concentration of SDS-PAGE being optimized. The results showed that when using 17 cm linear IPG strips pH ranged from 4 to 7, 11% SDS-PAGE, loading 180 μg/350 μl, proteins were better separated. Much more protein spots had been observed in 2-DE maps of the protein samples prepared by the PEG fractionation than TCA/acetone precipitation. In 2-DE profiles of the protein sample from mutant L638-y prepared by the PEG fractionation, as much as 1 235±6 protein spots were identified, 330 more spots than those by TCA/acetone precipitation method. By using the PEG fractionation, 190 differential protein spots were identified between the 2-DE maps of the mutant L638-y and wild type L638-g, 100 more spots than those by TCA/acetone
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2010年第9期1025-1032,共8页
Progress In Biochemistry and Biophysics
基金
国家油菜产业技术体系(nycytx-00505)
西北农林科技大学拔尖人才支持计划项目资助~~
关键词
芥菜型油菜
黄化突变体
叶片蛋白
蛋白质提取
双向电泳
Brassica Juncea L., chlorophyll-deficient mutant, leaves proteins, protein extraction, two-dimensionaleleetrophoresis
