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几乎是空气中的1万倍，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。这种二氧化碳浓缩机制对光合作用和藻类的生存至关重要。在光合作用的蓝藻中，二氧化碳浓缩机制包括将碳酸氢盐(HCO3-)从介质输送到细胞内的膜蛋白。然后，碳酸酐酶将HCO3-转化为CO2，由Rubisco固定，Rubisco是固定CO2的酶。真核藻类中的二氧化碳浓缩机制还不是很清楚，但已知涉及运输蛋白和多种碳水解酶。本项目的目标是研究真核绿藻-莱茵衣藻中二氧化碳的浓缩机制。模式单细胞生物C.reinhardtii是研究二氧化碳浓缩机制的理想生物，因为它的基因组是测序的，它具有强大的二氧化碳浓缩机制，并且已经发现了在二氧化碳浓缩机制方面存在缺陷的突变菌株。这些都是重要的工具，将被用来确定二氧化碳浓缩机制如何运作。提出了三项主要的试验性举措。第一组实验建立在先前研究的基础上，以进一步确定莱茵梭菌的碳酸氢酶基因家族的特征。通过生物信息学和经典生化研究，在莱茵梭菌中发现了9个不同的碳酸氢酶基因。这些蛋白质的表达和细胞内位置将被确定。RNA干扰(RNAi)将被用来减少特定的碳酸氢酶基因的表达，以确定它们是否参与了二氧化碳浓缩机制。该提案的第二个目标是识别和表征携带HCO3-的运输蛋白。在这些研究中，编码被认为是碳酸氢盐转运蛋白的基因将被引入一种蓝藻菌株，该菌株缺少所有已知的碳酸氢盐转运蛋白。第三个目标是鉴定叶绿体类囊体和叶绿体被膜上的转运蛋白。在这些研究中，蛋白质组学将被用来识别可能是二氧化碳浓缩机制组成部分的蛋白质。这项工作将增加对二氧化碳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二氧化碳水平上升的反应。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)。拟议的研究将为这些教育项目提供一个框架。

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.