紫穗槐菌根蛋白双向电泳分离体系的构建

Construction of Two-Dimensional Electrophoresis System for Separated Mycorrhizal Proteins of Amorpha fruticosa Inoculated with Glomus mosseae

丛枝菌根(arbuscular mycorrhizal,AM)真菌与宿主植物紫穗槐形成菌根过程中,双方都会发生一系列复杂的形态学和生理学变化,是一个多方面参与并精细调控的信号事件。通过温室盆栽试验,运用蛋白质组技术研究了AM真菌侵染紫穗槐过程中产生的共生相关蛋白,采用改良酚提取法提取紫穗槐菌根蛋白,并对蛋白上样量、染色方法等进行优化。结果表明,采用改良酚提取法,选用24 cm pH值4～7的胶条、1 000μg上样量、考马斯亮蓝R-350染色可获得分辨率高、重复性好的双向电泳图谱;应用凝胶分析软件Image Master TM 2D Platinum(Version 7.0)分析紫穗槐AM真菌侵染率达到90%时的菌根蛋白,接种AM真菌处理的GM组共获得434个蛋白点,而作为对照的CK组共获得419个蛋白点。其中GM组蛋白点与CK组相比较,上调表达蛋白点14个,下调表达蛋白点3个,特异表达蛋白点15个。

A pot culture experiment was carried out in greenhouse, and proteomics technology was used to study the symbiosisrelated proteins between AM fungi and Amorpha fruticosa. Total root proteins of A. fruticosa were extracted by optimized phenol-extracted method, and then the loading amount and staining method were optimized. Results show that high-resolu- tion reproducible two-dimensional eleetrophoresis （2-DE） gels could be obtained by the optimized phenol-extracted method, using 24 em IPG strip with a linear gradient of pH 4-7 for IEF, and protein samples loaded with 1 000 mg, visualized by Coomassie brilliant blue R-350. Analyzed with Image Master TM 2D Platinum software （ Version 7.0）, the 2-DE image of A. fruticosa root proteins inoculated with Glomus mosseae under a colonization rate of up to 90% exhibited 400 protein spots, among which 14 protein spots were up-regulated, 3 protein spots down-regulated, and 15 uniquely expressed.