杜氏盐藻完整叶绿体的分离及其蛋白提取  

Isolation of Intact Chloroplasts and Comparison of Methods for Its Protein Extraction from Dunaliella salina

作　　者：贾岩龙[1] 柴玉荣[1] 曲东京[1] 刘红涛[1] 薛乐勋[1] 

机构地区：[1]郑州大学细胞生物研究室郑州大学第一附属医院,郑州450052

出　　处：《生物技术通报》2008年第3期135-138,共4页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(No30540067)

摘　　要：应用高压破碎及蔗糖密度梯度离心的方法,分离出杜氏盐藻的完整叶绿体,随后用冻融法和研磨法分别提取叶绿体蛋白,并通过蛋白定量和SDS-PAGE凝胶电泳对两种蛋白提取方法进行比较,确立了一套适用于杜氏盐藻叶绿体分离和蛋白提取、定量以及电泳的方法。结果表明:用高压破碎结合蔗糖密度梯度离心的方法能够获得完整且较纯的盐藻细胞叶绿体;SDS-PAGE凝胶电泳结果显示,冻融法提取叶绿体蛋白效率高,电泳条带清晰,蛋白数量较多,蛋白齐全。为下一步在亚细胞水平进行杜氏盐藻耐盐机制的蛋白组学研究奠定了基础。In order to clarify the molecular basis for salinity tolerance in halotolerant green alga Dunaliella salina, isolation of intact chloroplasts and extraction of the protein from the alga are critical, In this study,cells of Dunaliella salina were broken using cell disruption by Nitrogen decompression and the chloroplasts were isolated by centrifugation through sucrose gradient. Then,two kinds of methods for protein extraction,freezing-thawing cycles and grinding,were compared to determine their efficiency in separating chloroplast proteins by concentration determination and SDS-PAGE gel electrophoresis. The results indicate that it was an efficient method of isolating intact chloroplasts using cell disruption by Nitrogen decompression and centrifugation through sucrose gradient. These results suggest that the method for isolating intact chloroplast and protein extraction are efficient and reliable for further 2-D gel electrophoresis of chloroplast proteins and identification by mass spectrometry,thus paved the way for dissecting molecular mechanisms of salinity tolerance in Dunaliella salina.

关 键 词：杜氏盐藻 叶绿体 蛋白提取 SDS—PAGE 

分 类 号：Q943.2[生物学—植物学]