苹果树腐烂病菌胞外蛋白提取方法  

Study on Extracting of Extracellular Proteins in Valsa mali

作　　者：王海英[1,2] 何媛媛[2,3] 黄丽丽[1,2] 韩青梅[1,2] 

机构地区：[1]西北农林科技大学植物保护学院,杨凌712100 [2]旱区作物逆境生物学国家重点实验室,杨凌712100 [3]西北农林科技大学理学院,杨凌712100

出　　处：《生物技术通报》2014年第7期49-53,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31171796);高等学校博士学科点专项科研基金(20120204110002);高等学校学科创新引智计划(B07049);公益性行业(农业)科研专项(201203034)

摘　　要：选择一种理想的蛋白提取方法,获得数量多、质量高的苹果树腐烂病菌Valsa mali胞外蛋白用于蛋白质组学分析,为全面解析该病原菌的致病机制奠定基础。采用冷冻干燥透析结合法、脱氧胆酸钠(DOC)-10%TCA沉淀法、硫酸铵沉淀法3种方法分别提取苹果树腐烂病菌的胞外蛋白,并筛选最适的硫酸铵浓度,利用Bradford法测定蛋白总量、SDS-PAGE检测蛋白条带分辨率及不同致病力菌株蛋白差异。结果表明,70%硫酸铵沉淀提取的胞外蛋白量最高、SDS-PAGE电泳可辨认蛋白条带最多。强弱致病菌株胞外蛋白的SDS-PAGE电泳图谱在37-50 kD有4条蛋白条带差异明显,由此表明,70%硫酸铵盐析法更适合用于苹果树腐烂病菌胞外蛋白质差异分析时蛋白质的提取。An optimal protein extraction method, as much as possible to obtain high-quality extracellular proteins of Valsa mali, is helpful to thoroughly analyze the proteomics and pathogenesis of the pathogen. In this study, the three methods freeze drying system/dialysis, DOC-10%TCA precipitation and ammonium sulfate precipitation were used to extract the extracellular proteins of Valsa mali, respectively. Then the optimal concentration of ammonium sulfate needed to be screened. The proteins extracted by the three methods were quantified and separated separately through Bradford and SDS-PAGE, and then extracellular proteins extracted from pathogenicity of different isolates were also compared with SDS-PAGE. The results showed that the content and distinguishability of protein sample extracted by 70%ammonium sulfate was optimal, and four protein lanes are significantly different in the SDS-PAGE electrophoresis patterns of different isolates. So this method is the most suitable for extracting extracellular protein sample to analyse the difference of Valsa mali protein.

关 键 词：苹果树腐烂病菌 硫酸铵沉淀 蛋白定量 聚丙烯酰胺凝胶电泳 差异蛋白 

分 类 号：S436.611[农业科学—农业昆虫与害虫防治]