Characterization of the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii

水生光合生物几乎占地球二氧化碳固定和氧气释放的50%。藻类对二氧化碳的固定在全球碳循环中起着重要作用，海洋中的大多数光合作用是由单细胞光合生物浮游植物完成的。 二氧化碳在空气中的扩散速度几乎是在水中的10，000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。 CO2浓缩机制对于大气CO2水平下的光合作用和浮游植物的生存至关重要。在这项研究中，衣原体reinhardtii将被用来确定二氧化碳浓缩机制的组成部分。C.将使用莱茵衣藻作为实验生物体，因为它是具有单个叶绿体的简单单细胞绿色衣藻。C.莱茵衣藻还具有强大的CO2浓缩系统，并且可以使用CO2作为碳源进行光自噬生长或使用乙酸盐作为碳源进行异养生长。对C.将使用插入诱变方法研究Reinahrdtii。插入诱变允许直接分离与突变表型相关的基因，从而允许功能的遗传和生化分析。在我们实验室以前的工作中，通过转化C. Reinhardtii细胞与BleR，一个基因编码抗生素Zeocin抗性。 在这些突变体中，不能生长在低CO2浓度，我们假设插入的BleR基因已经破坏了一个基因的CO2浓缩机制。 我们现在建议这些突变体的生理和遗传特征，并确定中断的基因。 将测试表现出BleR插入物与低CO2下生长不良的连锁的突变体的CO2固定和无机碳积累。一旦插入C.在reinhardtii基因组中，BleR提供了允许使用反向PCR或TAIL-PCR的独特序列，并且这些方法将用于鉴定中断的基因。预期一些插入将在编码将CO2递送至固定CO2的酶（核酮糖-1，5-二磷酸羧化酶/加氧酶）所需的转运蛋白或碳酸酐酶的基因中。预计编码参与光呼吸和低CO2信号转导途径的蛋白质的基因也将被破坏。 CO2浓缩机制的蛋白质的表征将增加我们对藻类如何提高其CO2固定效率的理解。对这一过程的理解也将有助于科学家预测海洋生物如何应对全球二氧化碳浓度的增加。 这项研究也将有助于培养本科生和研究生。 研究生将学习分子生物学方法，并将这些方法应用于藻类如何获得二氧化碳进行光合作用的生理生态问题。 本科生研究人员也将为这个项目做出贡献，获得宝贵的研究经验。 我们的研究项目吸引了一些来自代表性不足群体的本科生，我们将继续从这些群体中招募。

Aquatic photosynthetic organisms account for almost 50% of the Earth's CO2 fixation and O2 evolution. The fixation of CO2 by algae plays an important role in the global carbon cycle, and most photosynthesis in the ocean is accomplished by phytoplankton, single-celled photosynthetic organisms. Carbon dioxide diffusion is almost 10,000 times faster in air than in water and almost all phytoplankton and macroalgae have a mechanism that concentrates CO2 from the environment for photosynthesis. The CO2 concentrating mechanism is essential for photosynthesis at atmospheric levels of CO2 and for the survival of phytoplankton. In this study, Chlamydomonas reinhardtii will be used to identify components of the CO2 concentrating mechanism. C. reinhardtii will be used as the experimental organism since it is a simple unicellular green alga with a single chloroplast. C. reinhardtii also possesses a robust CO2 concentrating system and can be grown photoautophically using CO2 as the carbon source or heterotrophically using acetate as the carbon source. The CO2 concentrating mechanism of C. reinahrdtii will be studied using an insertional mutagenesis approach. Insertional mutagenesis allows for the direct isolation of genes associated with a mutant phenotype, thus permitting a genetic and biochemical analysis of function. In previous work from our laboratory, a large number of mutants that cannot grow on low concentrations of CO2 were generated by transforming C. reinhardtii cells with BleR, a gene encoding resistance to the antibiotic Zeocin. In these mutants that cannot grow on low CO2 concentrations, we hypothesize that the inserted BleR gene has disrupted a gene essential to the CO2 concentrating mechanism. We now propose to characterize these mutants physiologically and genetically and identify the interrupted genes. Mutants exhibiting linkage of the BleR insert to poor growth on low CO2 will be tested for CO2 fixation and inorganic carbon accumulation. Once inserted into the C. reinhardtii genome, BleR offers unique sequences allowing the use of inverse PCR or TAIL-PCR and these methods will be used to identify the interrupted gene. It is expected that some of the insertions will be in genes encoding transport proteins or carbonic anhydrases required for the delivery of CO2 to the enzyme that fixes CO2, ribulose-1,5-bisphosphate carboxylase/oxygenase. It is also expected that genes encoding proteins involved in photorespiration and the low CO2 signal transduction pathway will be disrupted. The characterization of the proteins of the CO2 concentrating mechanism will add to our understanding of how algae have increased their efficiency of CO2 fixation. An understanding of this process will also help scientists predict how marine organisms might respond to increasing global CO2 concentrations. This research will also contribute to the training of students at the undergraduate and graduate level. Graduate students will learn molecular biology methods and apply these methods to the ecophysiological question of how algae acquire CO2 for photosynthesis. Undergraduates researchers will also contribute to this project, gaining valuable research experience. Our research program has attracted a number of undergraduates from underrepresented groups and we will continue to recruit from these groups.