蓝藻prx基因家族成员功能研究

Peroxiredoxins (Prx) are central elements of the antioxidant defense system in the plant cells and cyanobacterial cells. The primary function of Prxs is working as thiol-dependent enzymes that decompose peroxides. Thus, they belong to the enzyme group of peroxidases. Prx proteins undergo major conformational changes in dependence of their redox state. Thus, they not only modulate cellular reactive oxygen species- and reactive nitrogen species dependent signaling, but depending on the Prx type they sense the redox state, transmit redox information to binding partners, and function as chaperone or molecular switch. The genome DNA of Cyanobacterium S. elongatus PCC 7942 encodes 6 different members of prx gene family. Under the stress triggered by the intracellular ROS boosting, the expression pattern of these different members of prx family exhibites a gene specific manner. Interestingly, the 2-Cys Prx, highly conserved antioxidant proteins, undergo 24 hour redox cycles, which persists for many days under constant light and temprature conditions. This phenomenon is quitely different from the molecular mechanism of current models of bioclock，the latter ,i.e "Central Clock" is based on the transcription-translation feedback loops(TTFL). This project will construct different Cyanobacterium cell strains with single gene knockout or double gene knockout of prx family members,then detect the different phenotype of individual knockout strains. Subsequently,we could give more reliable and valuable molecular evidences for identifying the function site of diferrent prx family member in performing their antioxidative function. Additionally, further research on the phenotype analysizing of these knockout strains,will confirm that whether there is a non-transcriptional oscillations of 2-Cys Prx which serves as a conserved rhythmic genetic marker persisting in the Cyanobacterium cells .These findings may facilitate more sophisticated cellular clock models, and highlight the interdependency of transcriptional and non-transcriptional oscillations in potentially all eukaryotic and prokaryotic cells.

peroxiredoxin（prx）基因家族的表达产物是植物和蓝藻细胞内抗氧化防御系统的核心因子。蓝藻S. elongatus PCC 7942 基因组共编码6个不同prx基因家族的成员，逆境胁迫条件下这些基因呈现出差异表达的特性。2-Cys Prx蛋白还能够保持周期大约为24小时的周期性氧化状态循环。这一现象与传统细胞"生物钟"分子机理，即"核心钟"元件依靠转录-翻译负反馈机制（TTFL）维持运转的模式完全不同。本项目拟分别构建蓝藻prx基因家族的单基因或双基因敲除细胞系，并对所有基因敲除系进行抗逆性表型分析，为确定prx基因家族的6个成员各自不同的功能定位寻找确凿的分子证据。如能确定蓝藻细胞内存在与真核细胞性质类似的不依赖于转录调控，由2-Cys Prx 氧化还原状态周期性变化组织的保守生物节律性遗传标签，将推进人们对于生物钟分子机制的认识，为进一步应用生物钟理论于生产实践打下基础。

Peroxiredoxin基因家族的表达产物是蓝藻细胞内抗氧化防御系统的核心因子。蓝藻S. elongatus PCC 7942共编码6个不同prx基因家族的成员，这些基因在不同生长条件下，呈现出差异表达的特性。其中prx-2cys基因的表达产物不仅能够清除细胞内的活性氧，而且还能在缺乏转录调控机制的情况下保持周期大约为24h的周期性氧化状态循环，而其它prx家族成员则尚未发现与之类似的非转录依赖节律性变化。这一现象与经典细胞生物钟分子机理，即“核心钟”元件感受环境信号后通过转录-翻译负反馈机制调钟控基因表达的生物钟运转模式完全不同。本项目以蓝藻Synechococcus elongatus PCC 7942为材料，分别构建了蓝藻prx基因家族的6个成员单敲除突变体及将prx-2cys和其它5个成员组合的双基因敲除突变体，进而对这些prx敲除系分别进行了活性氧胁迫条件下的表型分析。最终确定：prx基因家族不同成员的敲除细胞系与野生型相比处于显著的竞争劣势，其中prx-QA1 KO和prx-QA3 KO株系的生长速率最低。除prx-2cys基因外，prx家族的prx-QA1，prx-QA3以及prx-1cys基因也在蓝藻清除细胞内过量活性氧的过程中起作用，而prx-QA2，prx-QB等则不承担重要责任。prx-1cys和prx-2cys敲除株系还表现出与野生型蓝藻类似的H2O2胁迫抗性生长节律性。prx-1cys, prx-QB和prx-QA3可在prx-2cys缺失的条件下可以应激性地上调其表达水平，揭示了这些prx家族成员间存在代偿现象，而prx-QA1和prx-QA2则未发现类似的代偿作用。Western Blot的结果显示：prx家族成员的双敲除未对Kai生物钟节律性产生显著性影响，prx-2cys基因的缺失不能对Kai生物钟调控机制产生直接影响，而PRX-SO2/3节律性标签的产生与维持是翻译后修饰调控的结果，其调控过程不受TTFL影响。但prx-1cys、prx-QA1、prx-QA2等prx家族成员的缺失对Kai生物钟节律性的振幅等非主要参数产生影响，暗示了prx间接影响了Kai生物钟的表型。这些研究结果的获取为进一步明确PRX-SO2/3节律性遗传标记的分子调控机制和生理学意义的研究提供了直接和确凿的理论依据，具有重要的理论价值。