Reverse genetics of Chlamydomonas using TILLING

描述（由申请人提供）：项目概述：单细胞绿色衣藻莱茵衣藻是研究真核细胞生物学、生理学和生物化学许多基本方面的模式生物。例如，鞭毛/纤毛、基体和质体的生物发生和功能最容易使用衣原体进行研究。美国能源部联合基因组研究所已经公布了衣原体核基因组序列草案，覆盖率> 9倍，但缺乏反向遗传资源是目前衣原体功能基因组学的主要限制。TILLING是一种高通量反向遗传学方法，将传统诱变与现代技术相结合，用于检测点突变。TILLING可以在任何感兴趣的基因中提供一系列等位基因突变。这个项目的目标是建立一个公开可用的TILLING资源的衣原体研究社区，与西雅图TILLING项目，已成功地开发了类似的资源拟南芥，果蝇和斑马鱼社区合作。该提案的具体目的是（1）优化单倍体衣原体的UV/化学诱变并产生TILLING的突变群体，（2）进行试点TILLING项目以确定单倍体突变密度是否足以进行成本有效的TILLING，（3）优化二倍体衣原体的UV/化学诱变并产生TILLING的突变群体，（4）使用诱变的二倍体群体进行TILLING试验项目，（5）为衣原体研究团体提供TILLING服务。社区广泛支持开发衣原体TILLING资源。该项目产生的突变群体和数据将从衣原体库存中心和数据库公开提供。相关性：衣原体是基本生物学机制和人类疾病过程如多囊肾病、原发性纤毛运动障碍和Rh无效疾病的重要模型。这个项目将产生一个资源，使许多基因的基因功能的测定已经发现的衣原体基因组测序。

DESCRIPTION (provided by applicant): Project Summary: The unicellular green alga Chlamydomonas reinhardtii is the model organism of choice for studying many fundamental aspects of eukaryotic cell biology, physiology, and biochemistry. As examples, the biogenesis and function of flagella/cilia, basal bodies, and plastids are most easily investigated using Chlamydomonas. A draft sequence of the Chlamydomonas nuclear genome at >9X coverage has been released by the DOE's Joint Genome Institute, but the lack of reverse genetic resources is currently a major limitation for functional genomics of Chlamydomonas. TILLING is a high-throughput reverse genetics approach that combines traditional mutagenesis with modern technology for detection of point mutations. TILLING can provide an allelic series of mutations in any gene of interest. The goal of this project is to establish a publicly available TILLING resource for the Chlamydomonas research community, in collaboration with the Seattle TILLING Project, which has successfully developed similar resources for the Arabidopsis, Drosophila, and zebrafish communities. The specific aims of this proposal are (1) to optimize UV/chemical mutagenesis of haploid Chlamydomonas and generate mutant populations for TILLING, (2) to perform pilot TILLING projects to determine if the haploid mutation density is sufficient for cost-effective TILLING, (3) to optimize UV/chemical mutagenesis of diploid Chlamydomonas and generate mutant populations for TILLING, (4) to perform pilot TILLING projects using a mutagenized diploid population, and (5) to provide a TILLING service to the Chlamydomonas research community. There is broad community support for the development of a TILLING resource for Chlamydomonas. The mutant populations and data generated by this project will be publicly available from the Chlamydomonas stock center and database. Relevance: Chlamydomonas is an important model for basic biological mechanisms and for human disease processes such as polycystic kidney disease, primary cilia dyskinesia, and Rh null disease. This project will generate a resource that will enable the determination of gene function for many genes that have been discovered by sequencing the Chlamydomonas genome.