RAS-2调控粗糙脉孢菌生物钟的分子机制研究

Circadian clock is an important feature of organisms living on the earth, the oscillator of circadian clock can be regulated by multiple factors. The oscillator of Neurospora crassa is based on a feedback loop formed by WC-1, WC-2 and FRQ. The mechanism of RAS proteins regulating the circadian clock needs further investigation. RAS-2 protein regulates the circadian clock by targeting WC-1; RAS-2 activates protein kinase MAK-2, while WC-1 has potential phosphorylation sites of MAK-2. Based on these results, we would like to propose the following hypothesis to dissect the mechanism: RAS-2 activates MAK-2 kinase, which in turn phosphorylates WC-1 to regulate the transcriptional activity, further influence the expression of frq, thus regulating the circadian clock. We are going to mutate the phosphorylation sites of WC-1 to dissect the relationship between phsophorylation and transcriptional activity. We also study WC-1 phosphorylation and transcriptional activity, as well as frq transcription and conidiation rhythm in mak-2KO, dominant-active and dominant-negative RAS-2 strains. RAS proteins are highly conserved in higher eukaryotes; our work will certainly contribute to the research of circadian clock in higher plants and animals.

生物钟是生命的重要特征，其核心振荡器受诸多因素调控。粗糙脉孢菌核心振荡器由WC-1、WC-2及FRQ形成的负反馈调控环组成。RAS蛋白对粗糙脉孢菌核心振荡器有调控作用，但机制不清。前期研究发现WC-1是RAS-2的重要调控靶标，WC-1有潜在的蛋白激酶MAK-2磷酸化位点，RAS-2能激活MAK-2。结合文献分析，我们推测RAS-2通过激活MAK-2，磷酸化WC-1调节其转录调节功能，进而影响frq基因转录来调控粗糙脉孢菌生物钟。为证实上述推测，本项目拟采用定点突变技术研究WC-1的磷酸化与转录活性的关系；构建mak-2基因缺失突变体、RAS-2持续激活及持续钝化突变体，检测WC-1的磷酸化及转录活性改变，观察对frq基因转录及FRQ昼夜节律的影响，阐明RAS-2调控生物钟的分子机制。研究结果不仅对深入理解粗糙脉孢菌生物钟的调控机制有重要理论意义，且可为高等动植物生物钟研究提供有益借鉴。

粗糙脉孢菌是研究生物钟和光反应过程的模式生物。粗糙脉孢菌的光反应过程主要由WC-1及WC-2蛋白介导。粗糙脉孢菌生物钟核心振荡器由WC-1、WC-2及FRQ形成的负反馈调控环组成。粗糙脉孢菌编码有ras1和ras2基因。RAS1蛋白参与粗糙脉孢菌生物钟输出途径的调控，但RAS2在生物钟和光反应过程的作用及机制不清。我们发现粗糙脉孢菌ras2基因缺失突变体菌丝表现出明显的黄色表型，胡萝卜色素合成基因al-2，cao-2及ylo-1的表达异常。竞争性生长管结果显示ras2基因缺失导致分生孢子昼夜节律异常。此外，Western blot检测发现ras2基因缺失突变体中FRQ蛋白的昼夜节律发生明显改变，WC-1及WC-2蛋白的总量减少。这些结果说明RAS2参与调节光反应过程和生物钟。蛋白质降解实验发现ras2基因缺失突变体中WC-1，WC-2蛋白及FRQ蛋白的稳定性都下降。而磷酸化水平分析表明ras2基因缺失突变体中WC-1和WC-2蛋白均呈现出低磷酸化状态。这些结果说明RAS2蛋白通过影响WC-1，WC-2及FRQ的磷酸化修饰和稳定性来调控光反应过程及生物钟。研究还发现ras2基因缺失突变体中PKA的激活态降低，双突变体分析结果表明RAS2能通过影响cAMP-PKA信号通路调控生物钟和光反应过程。利用特异性的磷酸化抗体进行Western blot检测发现ras2基因缺失突变体中MAK-2信号通路的激活程度降低。MAK-2信号通路基因缺失也会导致生物钟分生孢子昼夜节律及WC-1，WC-2蛋白的磷酸化状态发生改变。这些结果说明RAS2通过cAMP-PKA信号通路和MAK-2信号通路，影响WC-1，WC-2及FRQ蛋白的磷酸化修饰状态和稳定性，从而调控生物钟和光反应过程。研究结果将为高等动植物中进一步研究RAS蛋白调控生物钟等过程的分子机制提供有益的借鉴和参考。