Analyses of the Chlamydomonas Reinhardtii Genome: A Model, Unicellular System for Analyzing Gene Function and Regulation in Vascular Plants

9975765 Grossman参与本项目的人员是亚瑟·格罗斯曼（PI，华盛顿卡内基研究所），Paul列费夫尔和Carloyn Silflow（明尼苏达大学联合教授），约翰·戴维斯（联合PI，爱荷华州州立大学），伊丽莎白哈里斯（共同PI，杜克大学），大卫干（共同PI，博伊斯汤普森研究所，康奈尔大学），和罗纳德戴维斯（Co-PI，斯坦福大学）.莱茵衣藻（Chlamydiumreinhardtii）是一种单细胞单倍体绿色植物，它已经并将继续成为阐明植物基本生物学过程的一个非常重要的模式系统。衣原体的实验对于光合作用过程的解剖特别重要，因为衣原体可以在固定碳的外源来源上快速生长，并且是唯一遗传上易处理的真核生物，其影响光合作用和碳同化的所有方面的突变是有条件的而不是致命的。应用复杂的全基因组方法研究衣原体将增加其作为光合功能和调节分析模型的实用性。具体地，作为第一个目标，将使用从在各种环境条件下生长的细胞分离的RNA产生标准化的衣原体cDNA文库。cDNA的3'和5'序列将有助于产生独特的EST组，这些EST组将排列在多聚赖氨酸包被的载玻片上。这些阵列将被用来检查全球基因表达的条件下，显着改变光合机构的活动和生物合成。微阵列还将用于分析那些光合作用突变体中的基因表达，这些突变体在本质上似乎是调节性的（以确定调节元件的潜在基因靶点）。此外，将产生大部分cDNA的全长序列，这将代表衣原体核基因组的几乎完整的编码能力。第二个目标是建立一个核基因组的部分物理图谱，该图谱与遗传图谱相匹配，目的是利用这些信息进行快速有效的基因图位克隆。作图研究将集中在核基因组的特定区域，这些区域以定义在光合作用中具有关键功能的基因的突变为中心。第三个目标是完成叶绿体基因组的测序，产生和分析缺失每个叶绿体开放阅读框的菌株，并使用含有每个叶绿体开放阅读框的微阵列来表征叶绿体基因在不同环境条件下的全局表达。微阵列技术也将被用来评估所有叶绿体mRNA的丰度在核突变体，表现出异常的质体基因表达的转录和转录后控制。在这项工作的过程中，将开发的许多信息和许多工具，将允许光合作用的研究，以达到一个全球性的层面，这是至关重要的阐明叶绿体功能的动态性质和参与调节叶绿体基因表达的机制。

9975765GrossmanThe personnel involved in this project are Arthur Grossman (PI, Carnegie Institution of Washington), Paul Lefebvre and Carloyn Silflow (Co-Pls, University of Minnesota), John Davies (Co-PI, Iowa State University), Elizabeth Harris (Co-PI, Duke University), David Stem (Co-PI, Boyce Thompson Institute, Cornell University), and Ronald Davis (Co-PI, Stanford University).Chlamydomonas reinhardtii, a unicellular haploid green alga, has been and will continue to be a very important model system for elucidating basic biological processes in plants. Experimentation with Chlamydomonas is particularly important for the dissection of photosynthetic processes since this alga can be grown rapidly on an exogenous source of fixed carbon and is the only genetically tractable eukaryote for which mutations that affect all aspects of photosynthesis and carbon assimilation are conditional rather than lethal. The application of sophisticated genome-wide methodologies to studies of Chlamydomonas will augment its utility as a model for the analysis of photosynthetic function and regulation. Specifically, as a first goal, normalized Chlamydomonas CDNA libraries will be generated using RNA isolated from cells grown under a variety of environmental conditions. The 3' and 5' sequences of the cDNAs will help generate unique sets of ESTs that will be arrayed onto polylysine coated slides. These arrays will be used to examine global gene expression under conditions that markedly alter the activity and biosynthesis of the photosynthetic apparatus. The microarrays will also be used to analyze gene expression in those photosynthetic mutants that appear to be regulatory in nature (to identify potential gene targets for regulatory elements). Furthermore, full-length sequences will be generated for most of the cDNAs, which would represent almost the complete coding capacity of the Chlamydomonas nuclear genome. A second goal is to establish a partial physical map of the nuclear genome that is aligned with the genetic map with the aim of using the information to perform rapid and efficient map-based cloning of genes. The mapping studies will focus on specific regions of the nuclear genome that are centered around mutations that define genes having critical functions in photosynthesis. A third goal is to complete the sequence of the chloroplast genome, generate and analyze strains that are deleted for each of the chloroplast open reading frames, and use microarrays containing each of the chloroplast open reading frames to characterize global expression of chloroplast genes under different environmental conditions. The microarray technology will also be used to evaluate the abundance of all chloroplast mRNAs in nuclear mutants that exhibit aberrant transcriptional and post-transcriptional control of plastid gene expression. Much of the information and many of the tools that will be developed during the course of this work will allow studies of photosynthesis to attain a global dimension, which is critical for elucidating the dynamic nature of chloroplast function and the mechanisms involved in regulating chloroplast gene expression.