Cellular acclimation and signal transduction of the green alga Chlamydomonas reinhardtii under anaerobic conditions

绿藻莱茵衣藻厌氧条件下的细胞驯化和信号转导

• 批准号: 235088911
• 负责人: Privatdozentin Dr. Anja Christine Hemschemeier
• 金额: --
• 依托单位: Arbeitsgruppe Photobiotechnologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/235088911?language=en
• 关键词: Cellular; acclimation; signal; transduction; green

In the absence of molecular oxygen (O2), aerobic organisms have to generate sufficient energy via the inefficient substrate level phosphorylation while adjusting growth to impaired biosynthetic pathways. The acclimation to anaerobiosis is facilitated by altered substrate- and electron fluxes as well as the differential expression of genes. This project aims at analyzing the response of the unicellular green alga Chlamydomonas reinhardtii to the stress condition of anaerobiosis on metabolic and transcriptional levels.Genome-wide transcript profiles from anaerobic C. reinhardtii cultures which we obtained by RNA-sequencing resulted in the identification of two candidate genes. One of the encoded proteins, THB8, belongs to the family of truncated hemoglobins. We could already observe that post-transcriptional silencing of the THB8-gene results in C. reinhardtii transformants which hardly grow under anaerobic conditions in the light and in which several anaerobic key genes are not properly expressed. In view of the phenotype of the transformants and a nitric oxide- (NO-) binding capacity of recombinant THB8 we postulate that THB8 is involved in an NO-based signaling transduction pathway. The expression patterns of the THB8-knockdown transformants indicate a connection between a proper THB8 expression and anaerobic induction of the CYG12 gene which encodes a heme/NO-binding guanylate cyclase. CYG12 was also outstanding in the above mentioned whole-genome transcript profiles.During this project, the postulated NO-based signaling cascade will be examined by determining the biochemical and biophysical features of THB8 and CYG12 as well as by analyzing the physiological and genetic characteristics of THB8- and CYG12-knockdown strains. The biochemical part of this project will focus on characterizing the heme groups, the binding of ligands such as NO and on identifying interaction partners of THB8 and CYG12. This project part will also include the analysis of a fermentative enzyme, the pyruvate:ferredoxin oxidoreductase PFR1. According to our RNA-Seq data, the regulation of the PFR1 gene differs significantly from other genes involved in fermentation. The biochemical and biophysical characteristics as well as the possible reactions catalyzed by PFR1 will hopefully elucidate its physiological role and thereby give clues to the reason for the different PFR1 gene expression.The physiological-genetic part of this study will focus on the characterization of physiology and (NO-dependent) gene expression upon anaerobiosis of the THB8- and yet to be created CYG12-knockdown transformants. A major part of this project will the generation of whole genome transcript profiles of the knockdown strains in order to identify affected gene groups.

在没有分子氧(O2)的情况下，好氧生物必须通过低效的底物水平磷酸化来产生足够的能量，同时调整生长以适应受损的生物合成途径。通过改变底物和电子通量以及基因的差异表达，促进了对厌氧菌的适应。本课题旨在从代谢和转录水平分析单细胞绿藻莱茵衣藻对厌氧胁迫的响应。通过RNA测序获得莱茵衣藻厌氧培养物全基因组转录图谱，鉴定出两个候选基因。其中一种编码蛋白THB8属于截短血红蛋白家族。我们已经观察到，THB8基因转录后的沉默导致了莱茵哈迪尔乳杆菌的转化体，这些转化体在光照下几乎不能在厌氧条件下生长，并且其中的几个厌氧关键基因没有得到正确的表达。根据转化子的表型和重组THB8与一氧化氮(NO-)的结合能力，我们推测THB8参与了一条基于NO的信号转导途径。THB8基因敲除转化子的表达模式表明，适当的THB8表达和编码血红素/非结合鸟氨酸环化酶的CYG12基因的厌氧诱导之间存在联系。在这个项目中，我们将通过测定THB8和CYG12的生化和生物物理特性，以及通过分析THB8和CYG12基因敲除菌株的生理和遗传特征，来研究假设的基于NO的信号级联。该项目的生化部分将侧重于表征血红素基团、NO等配体的结合以及确定THB8和CYG12的相互作用伙伴。本项目部分还将包括对一种发酵酶--丙酮酸：铁氧还蛋白氧化还原酶PFR1的分析。根据我们的RNA-Seq数据，PFR1基因的调控与其他参与发酵的基因有很大的不同。Pfr1的生化和生物物理特性以及可能的反应将有望阐明其生理作用，从而为解释Pfr1基因表达差异的原因提供线索。本研究的生理遗传学部分将重点研究THB8-和尚未建立的CYG12-敲除转化子在厌氧条件下的生理和(非依赖)基因表达的特征。该项目的一个主要部分将是生成基因敲除菌株的全基因组转录谱，以便识别受影响的基因组。