Components of the CO2 concentrating mechanism of Chlamydomonas reinhardtii

水生光合作用生物占地球二氧化碳固定和氧气释放的50%。然而，由于二氧化碳在水中的扩散速度几乎是空气中的10000倍，几乎所有的浮游植物和大型藻类都有一种从环境中浓缩二氧化碳进行光合作用的机制。水生光合作用生物对无机碳的吸收是一种主要的营养通量，在全球碳循环中起着重要作用。二氧化碳浓缩机制对于大气中二氧化碳水平的光合作用以及原核藻和真核藻的生存是必不可少的。在蓝藻中，已知的二氧化碳浓缩机制包括从介质中获得HCO3-的转运体和将积累的HCO3-转化为CO2以供Rubisco固定的碳酸酐酶。真核藻类中二氧化碳的浓缩机制还不是很清楚，但涉及到无机碳转运体和碳酸氢酶。这项提议的目标是描述真核藻类的二氧化碳浓缩机制。在这项工作中，模式生物莱茵衣藻将被用来识别二氧化碳浓缩机制的组成部分。莱茵哈蒂藻是一种单细胞真核绿藻，具有强大的二氧化碳浓缩系统。此外，还对莱茵毛虫基因组进行了测序，并鉴定了10,000多个基因。这项建议的具体目的是确定莱茵梭菌二氧化碳浓缩机制的新成分，并进一步确定已确定的转运蛋白和碳酸氢酶的特征。提出了四项主要的试验性举措。该提案的第一个目标是识别在从高二氧化碳条件切换到低二氧化碳条件后增加表达的新基因。以前已经发现，只有在低二氧化碳条件下才能合成二氧化碳浓缩机制。在这些实验中，将使用新构建的包含10,000多个基因的大阵列。任何在低二氧化碳条件下表达增加的基因都应该使用这个大阵列来检测。第二个实验是筛选和鉴定在二氧化碳浓缩机制中存在缺陷的插入突变体。这两个拟议的实验，微阵列分析和插入突变，旨在识别潜在编码二氧化碳浓缩机制组成部分的基因。第三组实验将进一步鉴定莱茵梭菌的碳酸氢酶基因家族。目前已经发现了8个不同的碳酸酐酶基因，分别命名为Cah1-Cah8，其中至少有一部分在二氧化碳浓缩机制中起着重要作用。拟议的工作将集中在Cah7和Cah8，这是最后发现的两个碳酸氢酶基因。这个项目已经进行了很多年。在最后一组实验中，RNA干扰或RNAi将被用来减少潜在编码二氧化碳浓缩机制中转运蛋白的基因的表达。如果一个基因在二氧化碳浓缩机制中很重要，那么减少该基因的表达可能会使细胞无法在空气中二氧化碳水平下生存。新发布的衣藻基因组将极大地促进拟议的研究。拟议的工作还将在研究生、本科生和高中生的教育中发挥作用，这些学生来自不同的背景。在路易斯安那州立大学，有许多项目来培养不同群体的本科生参与研究(NSF REU、HHMI等)，其中许多项目的学生都在PI的实验室进行了研究。拟议的研究将为这些教育项目提供一个框架。

Aquatic photosynthetic organisms account for 50% of the Earth's CO2 fixation and O2 evolution. However, since the diffusion of CO2 is almost 10,000 times slower in water than in air, almost all phytoplankton and macroalgae have a mechanism that concentrates CO2 from the environment for photosynthesis. The uptake of inorganic carbon by aquatic photosynthetic organisms is a major nutrient flux and plays a significant role in the global carbon cycle. The CO2 concentrating mechanism is essential for photosynthesis at atmospheric levels of CO2 and for the survival of prokaryotic and eukaryotic algae. In cyanobacteria, the CO2 concentrating mechanism is known to include transporters for acquiring HCO3- from the medium and a carbonic anhydrase that converts the accumulated HCO3- to CO2 for fixation by Rubisco. The CO2 concentrating mechanism in eukaryotic algae is less well understood but involves inorganic carbon transporters and carbonic anhydrases. The goal of this proposal is to characterize a CO2 concentrating mechanism from a eukaryotic alga.For this work, the model organism Chlamydomonas reinhardtii will be used to identify components of the CO2 concentrating mechanism. C. reinhardtii is a unicellular, eukaryotic green alga which possesses a robust CO2 concentrating system. In addition, the C. reinhardtii genome has been sequenced and over 10,000 genes have been identified. The specific aim of this proposal is to identify new components of the CO2 concentrating mechanism in C. reinhardtii and to further characterize the transporters and carbonic anhydrases that have already been identified. Four major experimental initiatives are proposed. The first aim of the proposal is to identify new genes that increase in expression after a switch from high to low CO2 conditions. It has previously been found that the synthesis of the CO2 concentrating mechanism occurs only under low CO2 conditions. For these experiments the newly constructed macroarray containing more than 10,000 genes will be used. Any gene that increases in expression under low CO2 conditions should be detected using this macroarray. The second experiment is to screen for and characterize insertional mutants that have defects in the CO2 concentrating mechanism. These two proposed experiments, the microarray analyses and insertional mutagenesis, are designed to identify genes that potentially encode components of the CO2 concentrating mechanism. The third set of experiments will further characterize the carbonic anhydrase gene family of C. reinhardtii. Eight different carbonic anhydrase genes designated Cah1-Cah8 have been discovered and at least some of these carbonic anhydrases play important roles in the CO2 concentrating mechanism. The proposed work will focus on Cah7 and Cah8 the last two carbonic anhydrase genes found. This project has been ongoing for a number of years. In the final set of experiments, RNA interference or RNAi will be used to decrease the expression of genes that potentially encode transporters involved in the CO2 concentrating mechanism. If a gene is important in the CO2 concentrating mechanism, then reducing the expression of that gene may render the cell unable to survive on air levels of CO2. The newly released Chlamydomonas genome will significantly facilitate the proposed research. The proposed work will also play a role in the education of graduate students, undergraduates and high school students from a wide diversity of backgrounds. At Louisiana State University, there are many programs to foster participation of a diverse group of undergraduates in research (NSF REU, HHMI, etc), and students from many of these programs have done research in the PI's laboratory. The proposed research will provide a framework for these educational programs.