板栗疫病菌致病性机理的双向凝胶电泳法研究

Study on the pathogenicity of Cryphonectria parasitica by 2-DE

关键词:  蛋白质提取  双向凝胶电泳  蛋白质组  板栗疫病菌

Key words:  protein extraction  2-DE  proteomics  Cryphonectria parasitica

摘要:

双向凝胶电泳技术是蛋白质组学研究的基础性技术平台。如何得到一张高质量的双向凝胶电泳图谱是进行后续研究的关键。为探索适用于板栗疫病菌可溶性总蛋白的最佳提取条件,从蛋白组学角度来探索板栗疫病菌致病性机理,比较了目前在丝状真菌中常用的两种蛋白质提取方法,制备的蛋白质样品经双向凝胶电泳后,在凝胶上呈现的蛋白质斑点的丰度和分布特点。结果表明,两种方法获得的蛋白质主要集中分布在pH4～7的范围内; TCA-丙酮沉淀法得到的图谱分辨率高但是蛋白质总量很少。裂解液-TCA-丙酮沉淀法得到的蛋白质总量较大,通过cleanup kit处理后图谱分辨率可以达到差异蛋白组的要求。随机提取几个银染蛋白点用MALDI-TOF MS/MS进行分析,可以得到高质量的肽质量指纹谱。表明该样品制备方法可以满足蛋白质鉴定的要求。

Abstract:

Comprehensive protein extraction from Cryphonectria parasitica is a key step to achieve high quality protein separation in two dimensional electrophoresis(2-DE). Two routine cellular total protein extraction methods were compared in order to determine an optimal one for filamentous fungi. Then the extracted total proteins were subjected to 2-DE,and the better extraction method was determined by the indexes of protein distribution and abundance on corresponding silver-stained gel. Data showed that most proteins were distributed in the pH ranging from 4 to 7; Buffer-TCA-Acetone got much more whole protein,which was treated with cleanup kit could be separated very well by 2-DE.But TCA-Acetone method precipitated total protein which is less although which shows fine differentiation among protein spots. So we consider that Buffer-TCA-Acetone is appropriate protocol for C.parasitica. Several silver -stained protein spots were picked for MALDI-TOF MS/MS to identify and high-quality PMF and MS/MS spectras were obtained. Buffer-TCA-Acetone is suitable sample preparation protocol for C.parasitica proteomics.