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

9975765Grossman参与该项目的人员有Arthur Grossman （PI，华盛顿卡内基研究所），Paul Lefebvre和Carloyn Silflow（合作，明尼苏达大学），John Davies（合作，爱荷华州立大学），Elizabeth Harris（合作，杜克大学），David Stem（合作，Boyce Thompson研究所，康奈尔大学）和Ronald Davis（合作，斯坦福大学）。莱茵衣藻（Chlamydomonas reinhardtii）是一种单细胞单倍体绿藻，已经并将继续成为阐明植物基本生物过程的一个非常重要的模式系统。衣藻实验对于光合作用过程的解剖尤为重要，因为这种藻类可以在外源固定碳上快速生长，并且是唯一一种遗传上可处理的真核生物，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。将复杂的全基因组方法应用于衣藻的研究将增加其作为分析光合功能和调控的模型的效用。具体来说，作为第一个目标，将使用从各种环境条件下生长的细胞中分离的RNA来生成标准化的衣藻CDNA文库。cdna的3‘和5’序列将有助于生成独特的est序列，这些序列将被排列在聚赖氨酸包被的载玻片上。这些阵列将用于检查在显著改变光合装置的活性和生物合成的条件下的全局基因表达。微阵列还将用于分析那些在自然界中似乎具有调节作用的光合突变体的基因表达（以确定调节元件的潜在基因靶标）。此外，大多数cdna将生成全长序列，这将代表衣藻核基因组的几乎完整的编码能力。第二个目标是建立与遗传图谱一致的核基因组的部分物理图谱，目的是利用这些信息进行快速有效的基于图谱的基因克隆。测绘研究将集中在核基因组的特定区域，这些区域以突变为中心，定义了在光合作用中具有关键功能的基因。第三个目标是完成叶绿体基因组序列，生成和分析每个叶绿体开放阅读框中缺失的菌株，并使用包含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。微阵列技术还将用于评估核突变体中所有叶绿体mrna的丰度，这些突变体表现出对质体基因表达的异常转录和转录后控制。在这项工作的过程中，将开发的许多信息和工具将使光合作用的研究达到一个全局的维度，这对于阐明叶绿体功能的动态性质和调控叶绿体基因表达的机制至关重要。

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.