蓝藻过氧化物氧还蛋白在非转录依赖的生物节律性调控机制中的功能研究

Organisms have evolved endogenous biological clocks as internal timekeepers to coordinate metabolic processes with the external environment. For the past decades, the transcriptional/translational feedback loops (TTFLs) between the products of rhythmically expressed genes have been considered to drive the cellular circadian rhythms, and to explain the majority of circadian outputs. But recently, some non-transcriptional processes were shown to be the primary contributors to circadian rhythmicity in cyanobacterium. Interestingly, some reports also have confirmed that an ancient non-transcriptional oscillator (NTO),the circadian rotation of redox states of peroxiredoxin (PRX) proteins, would exist in diverse cells such as metazoans, plants, a fungus, a bacterium, and even an archaeon. This modification of PRX has been thought to constitute the first marker of circadian rhythmicity shared by all organisms. These key findings have profound implications on our understanding of the evolution and the molecular basis of traditional cellular circadian timekeeping knowledge system. Some important scientific questions were encountered to the circadian clock researchers. As how does the NTO couple to the TTFL? And how does the PRX rhythmic marker work in the time-keeping mechanism, especially in Cyanobacterium? To find out the experimental and theoretical evidences to answer above questions, our project is planned to be carried out by two reversed strategies. Firstly, we will construct a series of period abnormal cyanobacterium Synechococcus elongatus PCC 7942 strains by hit-and-run site direct mutagenesis method. Each of these strain should harbor a different site-direct mutant Kai C gene, and exhibit either elongated period , or shorted period ,or disordered period phenotype, while the 2cys-prx gene coding PRX will keep intact in these mutants. The time course gene expression profile of 2cys-prx, and the clock phenotype of PRX sulfonylation cycles should be tested by Real-time RT-PCR and Western blot methods. Then reversely, we will hold on detecting the time course gene expression profiles, as well as the clock phenotypes of the intact KaiC and the key TTFL proteins of out-put pathway in the 2cys-prx knockout cyanobactrium strain，also by Real-time RT-PCR and Western blot methods. Via analyzing the comparable data obtained from the above-mentioned reverse experiments, we desire to collect enough evidences to explain the function of PRX rhythmic marker in Cyanobacterium time-keeping mechanism.

传统生物钟理论认为，节律性表达基因翻译产物间的转录/翻译负反馈抑制环（TTFL）是节律性产生和输出的关键。但近年来，人们发现非转录/翻译振荡器（NTO）才是生物节律性产生和维持的"源动力"。过氧化物氧还蛋白（PRX）氧化还原状态节律性是第一种被报道的跨物种保守的NTO 节律性标签，这一发现也带来了新的科学问题，即PRX标签 在细胞时间保持机制中的作用如何？为了回答这一问题，我们拟以蓝藻为研究对象，应用荧光实时定量RT-PCR 和Western blot 等方法在传统生物钟模型（以Kai蛋白家族为核心）调控蛋白发生突变的蓝藻细胞中检测PRX 节律性标签的维持状况；同时在PRX 节律性标签缺失突变体中检测Kai 生物钟的节律性的表型。通过这2 组反向实验，我们将尝试在PRX 节律性标签和Kai 生物钟TTFL 之间建立功能联系，并注释PRX 在蓝藻内源时间保持机制中的作用。

生物钟是生物体为了协调自身代谢过程与外部环境的周期性变化而进化出的内源时间控制机制。在过去40年中，人们证实节律性转录的钟基因及其表达产物共同构成的转录/翻译反馈环（TTFL）是生成生物钟昼夜节律的核心调控机制。但不同进化阶元生命体中的研究显示，内源生物钟的核心钟组件在进化上并不保守。而过氧化还原蛋白（PRX）氧化还原状态的昼夜节律性震荡，是一种不同于传统TTFL机制生物钟的非转录依赖节律性震荡（NTO）标签。该标签还是人们发现的第一种广泛存在于后生动物、植物、真菌和细菌等多种生命体内的通用节律性标签。 Prx-SO2/3标签的发现对解释生物钟系统的演化及其分子调控机制具有重大意义。为了回答“非转录依赖的NTO机制是如何与依赖于转录/翻译活动的TTFL机制互作的”等关键理论问题，项目组以蓝藻S.elongatus PCC7942为研究对象，借助Western blot 和基于基因敲除的经典节律性表型分析技术研究了Prx-SO2/3标签与依赖于TTFL的Kai生物钟的关联关系。结果显示：在蓝藻细胞中，prx基因的敲除只能对Kai 生物钟的节律性表型产生轻微影响。这表明prx基因家族的成员可以间接影响Kai基因的转录，但Prx-SO2/3节律性标签并未直接参与Kai生物钟TTFL的调控过程。这在理论上证实蓝藻Kai生物钟与Prx-SO2/3节律性标签在分子调控方面保持相互独立，但二者在核心钟基因在转录水平上存在cross-talking现象。另一方面，Kai生物钟存在与否，均不显著影响Prx-SO2/3标签的产生与维持。但KaiC 的某些突变能够对Prx-SO2/3标签的节律性表型产生显著影响。这在理论上暗示，尽管Kai生物钟与Prx-SO2/3标签的分子调控保持相互独立，但二者仍可能共用某些特异性的调控因子。项目组进一步的研究结果还显示，Prx-2-cys 参与到了环境时刻信息输入Kai生物钟的调控过程。该蛋白在Kai生物钟调控系统保持完整的情况下不影响Kai生物钟的表型，但在CikA缺失的情况下，可以部分代偿Kai生物钟依赖于CikA的环境信息主输入途径的功能。这一发现在理论上完善了蓝藻生物钟分子调控途径理论模型，打开了细胞内氧化还原稳态调控蓝藻生物钟环境时刻信号输入过程的理论研究新方向。