阿拉伯芥BPC家族參與晝夜節律調控之基因功能研究

Abstract

生理時鐘 (circadian clock) 是一種內生性計時器，它使生物體內的各種生理過程，包含新陳代謝及生理行為與外界晝夜變化同步，確保能隨著晝夜週期規律振盪，進而幫助生物體能在環境中有最好的適應性。植物個體無法遷徙躲避逆境，當遭逢突發的環境變化時會觸發必要的因應機制，可能影響原本的生理節律，在危機解除時，生理時鐘便可幫助改變的節律回歸原本的週期。阿拉伯芥中，植物特有的GAGA序列結合因子 (GAGA-motif binding factor) BASIC PENTACYSTEINE（BPC）基因家族參與了由晝夜節律控制的各種生理過程，包括根生長、下胚軸延長和生殖器官的形成。儘管如此，這些BPC轉錄因子在真核生物晝夜節律振盪系統中的作用卻未被研究。由於BPC的結合序列在多個時鐘基因的上游調節區域可被找到，我們欲在本研究中檢視BPC家族是否參與晝夜節律的調控。生理時鐘由一群具相互回饋調控關係的基因環環相扣而成。CIRCADIAN CLOCK ASSOCIATED 1（CCA1）是第一個被發現的時鐘基因，其表現形式常做為生理時鐘核心振盪系統 (core oscillator system) 運行的指標，我們於攜帶CCA1啟動子::冷光素酶報導基因的轉植株，分別誘導BPC家族不同成員的過量表現，檢測各BPC成員在植物細胞中對CCA1啟動子的作用，共檢測了第一類和第二類BPC亞家族成員。我們發現當誘導第一類的BPC3或第二類的BPC4及BPC6過量表現時，CCA1啟動子的活性明顯地受到抑制，而第一類BPC1、BPC2並未顯著改變CCA1啟動子的表現模式。儘管先前關於阿拉伯芥BPC基因家族的遺傳研究已經揭示了BPC3和BPC6之間的拮抗作用，我們發現BPC3、BPC4和BPC6對生理時鐘運行有類似的抑制功能。

Circadian clock is an endogenous timekeeper that synchronizes various physiological processes in planta to oscillate upon the day-night cycle. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), the first identified clock regulator among those genes interlocked by regulatory feedback loops of the circadian clock. The regulatory role of CCA1 in the core oscillator system makes this gene a decent example for revealing features of circadian clock operation. In Arabidopsis, the plant-specific GAGA-motif binding factor, BASIC PENTACYSTEINE (BPC) family, involves in various physiological processes those controlled by the circadian clock, including root growth, hypocotyl elongation and reproductive organ formation. Nevertheless, the roles of these BPCs in the eukaryotic circadian oscillator are still elusive. Upon that putative BPC-binding sites were identified at upstream regulatory regions of multiple clock genes, our study aimed at examining the role of BPC in the regulation of the circadian clock. Functional assays for BPC members of two subfamilies class I and class II were conducted by the induction of BPC ectopic expression in the transgenic line carrying promoter::luciferase construct of CCA1. We found that the induction of class I BPC3 or class II BPC4, BPC6 ectopic expression, significantly suppressed the activity of CCA1 promoter. Although, class I BPC1 and BPC2 did not notably alter the expression profile of CCA1 promoter. Even though the antagonistic function between BPC3 and BPC6 has been revealed by a previous genetic study on the BPC family. Our results indicated that BPC3, BPC4 and BPC6 have similar molecular functions for the negative regulation of the circadian clock.