Prime T A, Sherrier D J, Mahon P, Packman L C, Dupree P
Department of Biochemistry, Cambridge, UK.
Electrophoresis. 2000 Oct;21(16):3488-99. doi: 10.1002/1522-2683(20001001)21:16<3488::AID-ELPS3488>3.0.CO;2-3.
We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.
我们介绍了使用拟南芥愈伤组织培养作为一种系统，利用液体培养的愈伤组织对植物细胞器进行蛋白质组学分析。这种愈伤组织相对均匀、可重复且富含细胞质，能提供足够数量的细胞器用于蛋白质组学研究。利用二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（2-D SDS-PAGE）以及肽测序或质谱方法，建立了线粒体、内质网（ER）、高尔基体/前液泡区室和质膜（PM）标志物的数据库。通过Triton X-114相分离法，将愈伤组织中主要的膜相关蛋白鉴定为整合蛋白或外周蛋白。该数据库用于定义拟南芥愈伤组织质膜上的特定蛋白质。这个细胞器蛋白质数据库为未来鉴定新型植物蛋白的表达和定位奠定了基础。
