The Components of the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii

包括莱茵衣藻在内的9304662种莫罗尼单细胞藻类具有在体内浓缩二氧化碳的能力，使这些生物能够在低于大多数高等植物所能容忍的二氧化碳浓度的情况下进行光自养生长。这种二氧化碳浓缩机制是可诱导的；它只存在于二氧化碳限制条件下培养的细胞中。这个Ci(Ci=CO2+HCO3-+CO3-2)积累系统的一个重要方面位于叶绿体中。从低二氧化碳培养细胞中分离出的完整叶绿体比从高二氧化碳培养细胞中分离出的完整叶绿体具有积累Ci水平高得多的能力。在上一次拨款期间，莫罗尼博士已经确定了至少六种多肽，它们是在低二氧化碳条件下生长而专门诱导的，在高二氧化碳要求的菌株CIA-5中缺失。其中一种是位于周质间隙的碳酸氢酶，另一种是定位于叶绿体被膜的36 kDa多肽。其他诱导蛋白的位置和功能尚不清楚。在过去的一年里，一个适应低二氧化碳条件的莱茵哈迪尔乳杆菌细胞的cDNA文库已经构建完成。通过差异杂交，已经鉴定出四类重组噬菌体，它们含有在四类低二氧化碳条件下优先表达的基因。通过研究运输系统的组成和叶绿体在二氧化碳吸收中的作用，来研究莱茵梭梭的二氧化碳浓缩机制。莫罗尼博士将首先确定最近发现的低二氧化碳诱导基因的特征，并将这些基因与已知由低二氧化碳生长条件诱导的多肽联系起来。将针对由LacZ蛋白组成的融合蛋白产生抗体，该融合蛋白与诱导基因的产物相连。然后，这些抗体将被用来通过提案中概述的细胞器准备来定位莱茵梭菌细胞内的蛋白质。莫罗尼博士还将产生需要高二氧化碳的突变株，专注于只缺少一种可诱导多肽的菌株。人们希望最终将特定的基因与其基因产物以及缺乏该基因产物的菌株联系起来。对这一运输系统蛋白质的表征将增加我们对藻类低二氧化碳调节以及叶绿体在这种光合作用适应中的作用的理解。***

9304662 Moroney Unicellular algae, including Chlamydomonas reinhardtii, have the capacity to concentrate carbon dioxide internally, allowing these organisms to grow photoautotrophically at carbon dioxide concentrations lower than most higher plants can tolerate. This carbon dioxide concentrating mechanism is inducible; it is present only in cells that have been grown with limiting carbon dioxide conditions. An important aspect of this Ci (Ci = CO2 + HCO3- + CO3-2) accumulation system is located in the chloroplasts. Intact chloroplasts isolated from low carbon dioxide-grown C. reinhardtii cells retain the ability to accumulate Ci to much higher levels than intact chloroplasts isolated from high carbon dioxide-grown cells. During the last grant period, Dr. Moroney has identified at least six polypeptides that are specifically induced by growth on low carbon dioxide and are missing in the high carbon dioxide- requiring strain cia-5. One of these inducible proteins is a carbonic anhydrase located in the periplasmic space and another is a 36 kDa polypeptide localized to the chloroplast envelope. The locations of other inducible proteins and their functions are unknown. In the past year, a cDNA library to C. reinhardtii cells adapting to low carbon dioxide conditions has been constructed. By differential hybridization, four classes of recombinant phages have been identified that contain genes that are preferentially expressed under four classes low carbon dioxide conditions. It is proposed to study the carbon dioxide concentrating mechanism in C. reinhardtii by characterizing the components of this transport system and by delineating the role of the chloroplast in carbon dioxide acquisition. Dr. Moroney will first characterize the low carbon dioxide-inducible genes recently identified and connect these genes with the polypeptides known to be induced by low carbon dioxide growth conditions. Antibodies will be raised against fusion proteins consisting of t he lacZ protein linked to the product of the inducible gene. These antibodies will then be used to localize the proteins within the C. reinhardtii cell using organelle preparations outlined within the proposal. Dr. Moroney will also generate mutants that require high carbon dioxide, focussing on strains that are missing only one of the inducible polypeptides. It is hoped to eventually link a specific gene with its gene product and with a strain deficient in that gene product. The characterization of the proteins of this transport system will increase our understanding of the low carbon dioxide adjustment in algae and the role of the chloroplast in this photosynthetic adaptation. ***