Chloroplast Protein Transport in the Moss Physcomitrella Patens

叶绿体是负责光合作用的亚细胞区室，光合作用是利用阳光的能量将二氧化碳和水转化为糖和氧气的过程。光合作用是由叶绿体内的蛋白质复合体完成的，这些蛋白质复合体由核基因和叶绿体基因编码。 大约90%的叶绿体蛋白质实际上是在叶绿体外合成的，并且必须在它们组装成光合机构之前从细胞质输入。 这些蛋白质中的大多数通过位于叶绿体被膜中的一般转运机制进入叶绿体。 虽然这些易位蛋白的身份是已知的，但它们的具体作用现在才被阐明。 本申请提出检查被认为在包膜蛋白易位中起作用的两种蛋白质Toc64和基质Hsp70的功能。 实验将在苔藓小立碗藓中进行，小立碗藓是一种模式植物，其中引入的外源基因容易在细胞核中与同源内源基因重组，从而允许靶向基因敲除和替换。 同源重组的过程将用于消除Toc64和Hsp70基因，并将检查突变体植物的叶绿体成熟和发育缺陷。 叶绿体也将从突变体植物中分离出来，并用于旨在鉴定叶绿体输入机制缺陷的生化实验。 选择性地改善作物性状的努力必然包括将蛋白质靶向叶绿体，植物细胞的大部分代谢活动发生在叶绿体中。 因此，充分了解叶绿体输入蛋白质的机制，即拟议研究的主题，对于实现植物基因工程在改善人类健康和营养方面的全部潜力至关重要。 此外，这项研究将导致大量的研究生和本科生，博士后科学家的培训。

Chloroplasts are the subcellular compartment responsible for photosynthesis, the process that uses the energy of sunlight to convert carbon dioxide and water to sugar and oxygen. Photosynthesis is accomplished by complexes of proteins within the chloroplast, which are encoded by both nuclear genes and chloroplast genes. Approximately 90% of the chloroplast proteins are actually synthesized outside the chloroplast and must be imported from the cytoplasm prior to their assembly into the photosynthetic apparatus. Most of these proteins enter the chloroplast through a general translocation machinery located in the chloroplast envelope. While the identities of some of these translocation proteins are known, their specific roles are only now being elucidated. This application proposes to examine the function of two proteins thought to play a role in envelope protein translocation, Toc64 and stromal Hsp70. The experiments will be performed in the moss Physcomitrella patens, a model plant in which introduced foreign genes readily recombine in the nucleus with the homologous endogenous genes, allowing for targeted gene knockout and replacement. This process of homologous recombination will be used to the eliminate Toc64 and Hsp70 genes, and the mutant plants will be examined for defects chloroplast maturation and development. Chloroplasts will also be isolated from the mutant plants and used in biochemical experiments designed to identify defects in the chloroplast import machinery. Efforts to selectively improve crop traits will necessarily include targeting proteins to the chloroplasts, where much of the metabolic activity of a plant cell occurs. Thus a full understanding of the mechanisms through which chloroplasts import proteins, the subject of the proposed research, is crucial for realizing the full potential of plant genetic engineering on improving human health and nutrition. In addition, this research will result in the training of numerous graduate and undergraduate students, and postdoctoral scientists.