栅藻Scenedesmus sp.蛋白质提取方法优化及氨氮与硝态氮处理下蛋白质组表达差异  

An Optimized Protocol for Crush Cell and Isolation of Protein from Scenedesmus sp. and Ammonia Toxicity Analysis

作　　者：贾其坤[1,2] 梁青[1,2] 吴后波[1] 王广华[1] 吴华莲[1] 李涛[1] 王兵 向文洲[1] 

机构地区：[1]中国科学院南海海洋研究所,广州510301 [2]中国科学院大学,北京100049 [3]深圳海王药业有限公司,深圳518057

出　　处：《生物技术通报》2015年第11期125-130,共6页Biotechnology Bulletin

基　　金：国家科技计划项目(2013BAD10B04-1);广东省中国科学院全面战略合作项目(2012B091100268);海洋经济创新发展区域示范专项(SZHY2012-B01-003)

摘　　要：旨在寻找一种快速高效提取栅藻Scenedesmus sp.蛋白质的方法并研究氨氮对该藻的毒害机制。使用液氮研磨和超声细胞破碎进行细胞破碎,丙酮沉淀和TCA-丙酮沉淀法进行蛋白纯化,SDS-PAGE进行蛋白样品丰度检验,2-DE电泳技术对差异表达蛋白进行分析。结果显示,对于栅藻Scenedesmus sp.,液氮研磨破碎细胞效果明显优于超声细胞破碎法,前者细胞破碎完全,内容物充分外泄。TCA-丙酮沉淀纯化得到的蛋白样品不仅浓度高,而且丰度和纯度均显著优于丙酮沉淀法。氨氮毒害作用下栅藻Scenedesmus sp.蛋白差异表达分析结果显示85个表达降低的蛋白和25个表达升高的蛋白,这些蛋白涉及光合作用、二氧化碳固定、糖酵解、蛋白质合成及非正常折叠蛋白降解、细胞分裂和物质运输,以及自由基清除等途径。应用液氮研磨和TCA-丙酮沉淀法可快速高效地提取栅藻Scenedesmus sp.蛋白质,氨氮毒害作用涉及光合作用、脂肪酸合成、非正常折叠蛋白降解等多种代谢途径。Propose an optimized protocol for crush cell and isolation of protein from Scenedesmus sp. and study the ammonia toxicity of this algal with this protocol. Liquid nitrogen grinding and ultrasonic crushing method was used to disrupt the algae and acetone precipitation and TCA-acetone precipitation methods to extract and purify protein. Results showed that we found that the combination of liquid nitrogen grinding and TCA-acetone precipitation was effective and efficient as the cell crushed adequately and the protein sample was purified well and the protein amount and abundance was higher. The preliminary inquiry of ammonia toxicity to Scenedesmus sp. with two-dimensional gel electrophoresis（2-DE）technology found that 85 protein spots decreased and 25 protein spots increased as the algae cultured with ammonia compared with nitrate nitrogen. Those proteins have been identified as involved in photosynthesis, carbon dioxide fixation, glycolytic, protein synthesis and degradation of ubiquitinated protein, cell division and material transportation, and radical scavenging pathway.

关 键 词：双向电泳 栅藻 细胞破碎 蛋白纯化 氨氮毒害 

分 类 号：Q946.1[生物学—植物学]