Reverse genetics of Chlamydomonas using TILLING

描述(申请人提供)：项目摘要：单细胞绿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首选模式生物。例如，利用衣藻可以最容易地研究鞭毛/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已经公布了衣藻核基因组9X覆盖范围的序列草案，但目前缺乏反向遗传资源是衣藻功能基因组学的主要限制因素。TILLING是一种高通量的反向遗传学方法，它结合了传统的突变和现代技术来检测点突变。TILING可以在任何感兴趣的基因中提供一系列等位基因突变。该项目的目标是与西雅图耕作项目合作，为衣藻研究界建立一个公开可用的耕作资源，西雅图耕作项目已经成功地为拟南芥、果蝇和斑马鱼群体开发了类似的资源。这项建议的具体目的是(1)优化单倍体衣藻的紫外线/化学诱变，并产生用于耕作的突变群体，(2)进行试点耕作项目，以确定单倍体突变密度是否足以进行经济高效的耕作，(3)优化二倍体衣藻的紫外线/化学诱变，并产生用于耕作的突变群体，(4)利用诱变的二倍体群体进行试点耕作项目，以及(5)为衣藻研究界提供耕作服务。开发衣藻的耕作资源得到了社区的广泛支持。该项目产生的突变种群和数据将从衣藻种群中心和数据库公开获得。相关性：衣藻是基本生物学机制和人类疾病过程的重要模型，如多囊肾病、原发纤毛运动障碍和Rh缺失症。该项目将产生一个资源，能够确定通过对衣藻基因组测序而发现的许多基因的基因功能。

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.