拟南芥中氮营养与生物钟互馈调控分子机制研究

Endogenous circadian clock are almost ubiquitous among organisms from cyanobacteria to mammals and regulated by diverse environmental factors. As an important environmental cue, how nitrogen nutrient affect central circadian components is still lacking intensive study. In turn, circadian clock could regulate nitrogen metabolism, forming a feedback loop. However, the molecular mechanism of circadian rhythms appear in nitrate primary assimilation is still not much clear. Herein, we try to analyze the expression levels of central circadian components and circadian reporter genes using nitrogen signal mutants subject to different nitrogen condition, to discover the mechanism of nitrogen input into circadian clock on the molecular level. Then, in order to reveal how circadian clock regulate nitrate primary assimilation, we will detect the circadian rhythms of nitrate reductase, a key enzyme in nitrate primary assimilation using cca1 mutant. We also plan to investigate the binding and regulating of CCA1 on NR1 or NR2 by yeast one-hybrid, EMSA, ChIP, transient experiment and transgenic plants. The aim of this project is to demonstrate the mechanism of reciprocal regulation between nitrogen nutrient and circadian clock, which will provide evidence for plant nutrition input into endogenous circadian clock. Prospectively, it may provide theoretical basis for improving nitrogen use efficiency.

生物钟广泛存在于各种生物体内，而且受环境因子的调控。植物的氮养分作为外界环境条件，如何影响植物内源生物钟还缺少深入的研究。反过来，生物钟参与调控氮代谢，从而形成一个相互反馈调控的循环，但是对于生物钟调控氮初级同化的分子机制，尚缺乏系统研究。因此，本项目通过研究不同氮营养条件对生物钟中央振荡器基因及节律报告基因的影响，来探讨氮营养如何在输入途径调控生物钟；其次，利用cca1突变体研究硝酸盐初级同化途径中关键性酶硝酸还原酶（NR）的生物节律表达特征，并利用酵母单杂交、EMSA、ChIP、烟草叶片转化实验和转基因植株，研究生物钟中央振荡器CCA1对NR编码基因NR1和NR2转录调控的分子机理，来阐述生物钟参与调控硝酸盐的同化。从而，最终揭示氮营养和生物钟互馈调控机制，为植物的营养条件是生物钟输入途径提供有力证据，并且在实践上为提高作物氮利用率提供理论依据。

本项目首先通过研究不同氮营养条件对生物钟中央振荡器基因表达的影响， 发现NO3-和NH4+能够影响TOC1的表达形式，初步探讨了氮营养有可能在输入途径调控生物钟。其次， 研究发现硝酸盐初级同化途径中关键性酶硝酸还原酶（NR）的表达呈现生物节律特征，并在生物钟cca1突变体中发生变化，说明生物钟核心元件CCA1转录水平调控NR 编码基因NR1和NR2。我们利用EMSA和ChIP实验验证了CCA1能够直接结合到NR1和NR2启动子上。后续对于转基因植株材料的研究，有希望进一步明确生物钟中央振荡器CCA1对 NR1和NR2转录调控的分子机理。从而，最终揭示氮营养和生物钟互馈调控机制，为植物的营养条件有可能为生物钟输入途径提供证据，并且在实践上为提高作物氮利用率提供理论依据。