Identification of a Novel Gene, CIA6, Required for Normal Pyrenoid Formation in Chlamydomonas reinhardtii
Identification of a Novel Gene, CIA6, Required for Normal Pyrenoid Formation in Chlamydomonas reinhardtii
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DOI：
10.1104/pp.111.173922
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发表时间：
2011-06-01
期刊：
影响因子：
7.4
通讯作者：
Moroney, James V.
中科院分区：
文献类型：
--
作者：
Ma, Yunbing;Pollock, Steve V.;Moroney, James V.
莱茵衣藻具有CO2浓缩机制（CCM），使其能够在低CO2浓度下生长。在具有CCM的光合生物中看到的一个共同特征是Rubisco在细胞内的紧密包装。在许多真核藻类中，Rubisco定位于类核蛋白，类核蛋白是叶绿体内的电子致密结构。为了鉴定功能性CCM所需的基因，产生插入博来霉素抗性（Ble（R））突变体，并筛选其在高CO2条件（空气中5%CO2）下在基本培养基上生长，但在极低CO条件（空气中0.01%CO2）下仅缓慢生长或不生长。从这个筛选中鉴定出的一个突变体被命名为cia 6。生理学研究证实，cia 6在低水平的二氧化碳中生长不良，积累无机碳的能力受损。插入的Ble（R）破坏了编码与含有SET结构域甲基转移酶的蛋白质具有序列相似性的蛋白质的基因，尽管使用过表达的CIA 6的实验未能证明甲基转移酶活性。电子显微镜显示cia 6突变细胞的蛋白核高度紊乱。突变体的互补恢复了蛋白核，在低CO2条件下生长的能力，以及浓缩无机碳的能力。定量逆转录-聚合酶链反应数据从低CO2诱导时间过程实验表明，上调的几个CCM组件是缓慢的cia 6与野生型相比。使用蛋白质印迹在蛋白质水平进一步证实了这种缓慢诱导。这些结果表明，CIA 6是蛋白核形成所必需的，并进一步支持了蛋白核是C.莱因哈德氏菌
Chlamydomonas reinhardtii possesses a CO2-concentrating mechanism (CCM) that allows the alga to grow at low CO2 concentrations. One common feature seen in photosynthetic organisms possessing a CCM is the tight packaging of Rubisco within the cell. In many eukaryotic algae, Rubisco is localized to the pyrenoid, an electron-dense structure within the chloroplast. In order to identify genes required for a functional CCM, insertional Bleomycin resistance (Ble(R)) mutants were generated and screened for growth on minimal medium under high CO2 conditions (5% CO2 in air) but only slow or no growth under very low CO conditions (0.01% CO2 in air). One mutant identified from this screen was named cia6. Physiological studies established that cia6 grows poorly on low levels of CO2 and has an impaired ability to accumulate inorganic carbon. The inserted Ble(R) disrupted a gene encoding a protein with sequence similarity to proteins containing SET domain methyltransferase, although experiments using overexpressed CIA6 failed to demonstrate the methyltransferase activity. Electron microscopy revealed that the pyrenoid of cia6 mutant cells is highly disorganized. Complementation of the mutant restored the pyrenoid, the ability to grow under low-CO2 conditions, and the ability to concentrate inorganic carbon. Quantitative reverse transcription-polymerase chain reaction data from a low-CO2 induction time-course experiment demonstrated that the up-regulation of several CCM components is slower in cia6 compared with the wild type. This slow induction was further confirmed at the protein level using western blots. These results indicated that CIA6 is required for the formation of the pyrenoid and further supported the notion that the pyrenoid is required for a functional CCM in C. reinhardtii.