Aronsson Henrik, Jarvis R Paul
Department of Plant and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden.
Methods Mol Biol. 2011;774:281-305. doi: 10.1007/978-1-61779-234-2_17.
In vitro chloroplast protein import assays have been performed since the late 1970s, initially with plant species (e.g., pea and spinach) that readily provide an abundant source of starting material and also, subsequently, a good yield of chloroplasts for import assays. However, the sequencing of the Arabidopsis genome paved the way for an additional model system that is more amenable to genetic analysis, as a complement to the more biochemically orientated models such as pea and spinach. A prerequisite for this change was an efficient and reliable protocol for the isolation of chloroplasts for use in protein import assays, enabling biochemical approaches to be combined with the genetic potential of the plant. The method described here was developed as a rapid and low-cost procedure that can be accessed by everyone due to its simplicity. Despite its rapidity and simplicity, the method yields highly pure chloroplasts, and in addition works well with mutant plants that exhibit pale or chlorotic phenotypes. The protocol is also optimized for work with material from young plants (10-14 days old), when protein import is believed to be at its peak, and so plant growth can be conducted in vitro on Murashige and Skoog medium. The isolation method has been used not only for protein import assays, but also for proteomic analysis and further subfractionation studies.
自20世纪70年代末以来，人们一直在进行体外叶绿体蛋白导入实验，最初使用的植物物种（如豌豆和菠菜）能轻易提供丰富的起始材料来源，随后也能为导入实验提供大量叶绿体。然而，拟南芥基因组的测序为另一种更适合遗传分析的模型系统铺平了道路，作为对豌豆和菠菜等更偏向生化研究的模型的补充。这种转变的一个先决条件是要有一个高效可靠的叶绿体分离方案，用于蛋白质导入实验，使生化方法能够与植物的遗传潜力相结合。这里描述的方法是作为一种快速且低成本的程序开发的，因其简单性，每个人都可以采用。尽管该方法快速且简单，但能产生高度纯净的叶绿体，此外，对于表现出苍白或黄化表型的突变植物也适用。该方案还针对使用来自10至14日龄幼嫩植物的材料进行了优化，据信此时蛋白质导入处于高峰期，因此可以在Murashige和Skoog培养基上进行体外植物培养。该分离方法不仅用于蛋白质导入实验，还用于蛋白质组学分析和进一步的亚分级研究。
