The Carbon Dioxide Concentrating Mechanism of Chlamydomonas Reinhardtii

水生环境中的浮游植物和藻类占地球上通过光合作用固定的二氧化碳（CO2）的近50%。 由于二氧化碳在水中的扩散速度比在空气中慢近10，000倍，几乎所有这些浮游植物和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。 这种CO2浓缩机制对于光合作用和藻类的生存至关重要。在光合蓝藻中，CO2浓缩机制包括将重碳酸盐（HCO 3-）从培养基转运到细胞中的膜蛋白。 然后碳酸酐酶将HCO 3-转化为CO2，以通过Rubisco固定CO2。真核藻类中的CO2浓缩机制还不太清楚，但已知涉及转运蛋白和多种碳酸酐酶。 本项目的目标是表征存在于真核绿色衣藻莱茵衣藻中的CO2浓缩机制。 模式单细胞生物C. reinhardtii是研究CO2浓缩机制的理想菌株，因为基因组被测序，它具有稳健的CO2浓缩机制，并且已经鉴定出在CO2浓缩机制中具有缺陷的突变菌株。 这些都是重要的工具，将用于确定如何CO2浓缩机制的功能。 提出了三大试点举措。 第一组实验建立在以前的研究基础上，以进一步表征C.莱因哈德氏菌 通过生物信息学和经典生物化学的研究，在C.莱因哈德氏菌 将确定这些蛋白质的表达和细胞内定位。 RNA干扰（RNAi）将用于减少特定碳酸酐酶基因的表达，以确定它们是否参与CO2浓缩机制。 该提案的第二个目的是鉴定和表征携带HCO 3-的转运蛋白。 对于这些研究，编码被认为是碳酸氢盐转运蛋白的蛋白质的基因将被引入缺失所有已知碳酸氢盐转运蛋白的蓝藻菌株中。 第三个目标是确定叶绿体类囊体和叶绿体包膜膜上的转运蛋白。 对于这些研究，蛋白质组学将用于鉴定可能是CO2浓缩机制组分的蛋白质。 这项工作将增加对CO2浓缩机制如何促进全球碳循环的理解，并将有助于预测藻类对大气CO2水平上升的反应。 拟议的工作还将在来自各种背景的研究生、本科生和高中生的教育中发挥作用。 在路易斯安那州立大学，有许多项目，以促进参与研究的本科生的多元化群体（NSF REU，HHMI等）。 拟议的研究将为这些教育方案提供一个框架。

Phytoplankton and algae in aquatic environments account for almost 50% of the carbon dioxide (CO2) fixed by photosynthesis on the Earth. Because the diffusion of CO2 is almost 10,000 times slower in water than in air, almost all of these phytoplankton and algae have a mechanism that concentrates CO2 from the environment for photosynthesis. This CO2 concentrating mechanism is essential for photosynthesis and the survival of algae. In photosynthetic cyanobacteria, the CO2 concentrating mechanism includes membrane proteins that transport bicarbonate (HCO3-) from the medium into the cell. A carbonic anhydrase enzyme then converts the HCO3- to CO2 for fixation by Rubisco, the enzyme that fixes CO2. The CO2 concentrating mechanism in eukaryotic algae is less well understood but is known to involve transport proteins and multiple carbonic anhydrases. The goal of this project is to characterize the CO2 concentrating mechanism present in the eukaryotic green alga Chlamydomonas reinhardtii. The model unicellular organism, C. reinhardtii, is ideal for studying the CO2 concentrating mechanism because the genome is sequenced, it has a robust CO2 concentrating mechanism and mutant strains that have defects in the CO2 concentrating mechanism have been identified. These are important tools that will be used to determine how the CO2 concentrating mechanism functions. Three major experimental initiatives are proposed. The first set of experiments builds on previous studies to further characterize the carbonic anhydrase gene family of C. reinhardtii. Through bioinformatics and classical biochemical studies, nine different carbonic anhydrase genes have been discovered in C. reinhardtii. The expression and the intracellular location of these proteins will be determined. RNA interference (RNAi) will be used to reduce the expression of specific carbonic anhydrase genes to determine whether or not they are involved in the CO2 concentrating mechanism. The second aim of the proposal is to identify and characterize transport proteins that carry HCO3-. For these studies, the genes encoding proteins thought to be bicarbonate transporters will be introduced into a cyanobacterial strain that is missing all known bicarbonate transporters. The third aim is to identify transport proteins on both the chloroplast thylakoid and chloroplast envelope membranes. For these studies proteomics will be used to identify proteins likely to be components of the CO2 concentrating mechanism. This work will increase the understanding of how the CO2 concentrating mechanism contributes to the global carbon cycle and will facilitate the prediction of algal responses to rising atmospheric CO2 levels. 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). The proposed research will provide a framework for these educational programs.