Development of an In Vitro Assay for Chloroplast Protein Targeting in the Moss, Physcomitrella patens

叶绿体中大约90%的蛋白质在细胞核中编码，在细胞质核糖体上翻译，并在细胞分裂后穿过被膜输入。 这些蛋白质的一个大的子集被进一步靶向众多的内部质体隔室之一。 我们对这一过程的了解大多来自于离体叶绿体和类囊体的体外实验。 最近，已经描述了相对少量的突变体，其在质体蛋白质输入中是有缺陷的，允许输入过程的遗传分析。 然而，在同一植物中很难将遗传分析和生化分析联合收割机结合起来，因为便于遗传使用的生物化学对象较差，反之亦然。 苔藓小立碗藓在陆地植物中是独特的，其能够通过同源重组整合引入的DNA。 这为通过定点突变和等位基因置换来检测植物功能提供了可能。 如果该模式植物系统被证明是用于生化和遗传分析的良好生物体，则其实用性将显著增强。 本项目的目标是收集所需的工具，进行体外分析的叶绿体蛋白质靶向在苔藓，然后优化程序分离蛋白质进口主管叶绿体和类囊体。 希望这项工作将导致第一个植物模型的发展，其中生化和遗传工具可以在同一有机体中毫无困难地结合起来。

Approximately 90% of the proteins in chloroplasts are encoded in the nucleus, translated on cytoplasmic ribosomes and imported post-translationally across the envelope membranes. A large subset of these proteins are further targeted to one of the numerous internal plastidic compartments. Most of our knowledge of this process comes through in vitro experiments performed with isolated chloroplasts and thylakoids. More recently, a relatively small number of mutants have been described which are defective in plastid protein import, allowing genetic analysis of the import process. However, it has been difficult to combine both genetic and biochemical analyses in the same plant, as those that are convenient for genetic use are poor biochemical subjects, and vice versa. The moss Physcomitrella patens is unique among land plants in its ability to incorporate introduced DNA by homologous recombination. This opens the possibility to examine plant functions by site-directed mutation and allele replacement. The utility of this model plant system would be significantly enhanced if it were to prove to be a good organism for biochemical as well as genetic analysis. The objectives of this project are to collect the tools required to perform in vitro analysis of chloroplast protein targeting in the moss, and then to optimize the procedures for isolation of protein import-competent chloroplasts and thylakoids. It is hoped that this work will lead to the development of the first plant model in which biochemical and genetic tools can be combined without difficulty in the same organism.