Reverse genetics of Chlamydomonas using TILLING

项目概述：莱茵衣藻（Chlamydomonas reinhardtii）是研究真核细胞生物学、生理学和生物化学许多基础方面的首选模式生物。例如，用衣单胞菌最容易研究鞭毛/纤毛、基体和质体的生物发生和功能。美国能源部联合基因组研究所（Joint genome Institute）已经发布了覆盖范围为bbb90x的衣藻核基因组草图序列，但缺乏反向遗传资源是目前衣藻功能基因组学的主要限制。TILLING是一种高通量的反向遗传学方法，将传统的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等位基因系列突变。该项目的目标是与西雅图TILLING项目合作，为衣藻研究界建立一个公开可用的TILLING资源，西雅图TILLING项目已经成功地为拟南芥、果蝇和斑马鱼社区开发了类似的资源。本课题的具体目标是：(1)优化单倍体衣藻的紫外/化学诱变并产生TILLING突变群体，(2)进行中试TILLING项目，以确定单倍体突变密度是否足以进行高成本效益的TILLING，(3)优化二倍体衣藻的紫外/化学诱变并产生TILLING突变群体，(4)利用诱变的二倍体群体进行中试TILLING项目。(5)为衣藻研究界提供TILLING服务。衣藻TILLING资源的开发得到了广泛的支持。该项目产生的突变种群和数据将从衣藻库存中心和数据库中公开提供。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 null病）的重要模型。该项目将产生一个资源，将使确定基因功能的许多基因，已发现测序衣藻基因组。

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.