白叶枯病菌胞内蛋白的提取及电泳分离方法的构建  

Establishment of Extraction and 2-D Electrophoresis of Intracellular Protein of Xanthomonas oryzae pv.oryzae

作　　者：于海天[1,2] 阿新祥[2] 刘自单 张恩来[2] 汤翠凤[2] 杨雅云[2] 董超[2] 张斐斐[2] 陈丹[2] 陈丽娟[1] 戴陆园[2] 

机构地区：[1]云南农业大学,昆明650091 [2]云南省农业科学院生物技术与种质资源研究所,云南省农业生物技术重点实验室,农业部西南作物基因资源与种质创制重点实验室,农业部云南稻种资源科学观测实验站,昆明650223

出　　处：《基因组学与应用生物学》2015年第1期195-202,共8页Genomics and Applied Biology

基　　金：国家自然科学基金(31160274);云南省自然科学基金(2013FZ147);云南省农科院院专项基金(YAAS2013JC009,YAAS2013ZY011)共同资助

摘　　要：水稻白叶枯病菌已成为研究植物-病原物互作模式的病菌。要利用蛋白质组学方法深入研究其生物学特性和致病机理,人们面临着其胞内蛋白提取和有效分离方法尚未建立的问题。本文采用TCA丙酮沉淀法和裂解液沉淀法提取白叶枯病菌胞内蛋白,利用两种等点聚焦程序(IEFⅠ,IEFⅡ)进行一向等电聚焦,用三种蛋白上样浓度(400μg,600μg,800μg)进行双向电泳,G-250烤染后,通过Imagemaster软件对双向电泳图谱进行比较分析。结果表明,裂解液沉淀法、等电聚焦程序Ⅱ(IEFⅡ)、600μg上样浓度三者相结合的方法是提取和分离白叶枯病菌胞内蛋白的理想方法,能获得较好的实验效果。该方法的建立为白叶枯病菌蛋白质组学研究奠定基础。Rice bacterial blight（Xanthomonas oryzae pv.oryzae） has become an important mode of pathogenic bacteria in study of plant-pathogen interaction.In order to study the biological characteristics and the pathogenetic mechanisms of the Xanthomonas oryzae pv.oryzae,people faced the problem that the effective methods of extracting and isolating the intracellular protein have not been established yet.In this study,intracellular proteins of Xanthomonas oryzae pv.oryzae were extracted through TCA-DOC precipitation and lysis-buffer precipitation methods,two kinds of IEF system（systemI,systemⅡ）,three kinds of loading protein concentration（400 μg,600 μg,800 μg） were designed.After 2-D electrophoresis,G-250 dyeing,the 2-DE maps were analyzed with Imagemaster software.The results showed that the combination of lysis-buffer method,systemⅡ and loading protein concentration of 600 μg was more effective for intracellular protein extraction ofXoo.The establishment of this method might be practical for further study of the Xoo proteomics.

关 键 词：白叶枯病菌 胞内蛋白提取 双向电泳 分离方法 

分 类 号：S432.4[农业科学—植物病理学]