阿拉伯芥生長發育調節因子BASIC PENTACYSTEINE與生理時鐘調節因子LIGHT-REGULATED WDs之交互作用

Abstract

生活在地球上的生物透過接收外界的晝夜節律同步自身的計時器，生理時鐘，以維持生物之生長、發育、代謝等生理現象。已知模式植物阿拉伯芥中生理時鐘為基因的調節反饋迴圈構成，形成複雜的網路。近年研究發現，LIGHT-REGULATED WD1 (LWD1) 藉由與轉錄因子形成蛋白質複合體調控生理時鐘相關基因之啟動子，已知LWD1為時鐘基因CIRCADIAN CLOCK ASSOCIATED1 (CCA1)啟動子之正向調節因子。實驗室過往研究已知LWD1與BASIC PENTACYSTEINE (BPC)家族成員在轉錄層級上有共表現的現象，而BPCs為植物發育不可或缺的調節因子。已知植物在BPC缺失導致嚴重的發育表現型，且過表現BPC3會抑制CCA1的表現。然而BPCs與LWD1在轉錄層級上的共表現是否與BPCs參與生理時鐘調控有關並不清楚。為了進一步探討BPC家族成員是否能與LWD1共同調控生理時鐘基因的表現，我的研究目標為揭示BPCs與LWD1在細胞中交互作用的關係。首先我以分子遺傳方式測試BPC家族成員缺失對黎明基因CCA1與傍晚基因GIGANTEA (GI) 啟動子表現之影響，我發現CCA1與GI在bpc4 bpc6、bpc1 bpc2和bpc1 bpc2 bpc3中皆有表現減弱的情況，顯示BPCs成員可參與生理時鐘的正向調控。接著我藉由雙分子螢光互補 (BiFC)實驗測試，與實驗室前人發現BPC3、BPC4與LWD1具交互作用結果一致，我亦證實BPC1、BPC2、BPC6能與LWD1交互作用，顯示BPCs可能與LWD1共同參與生理時鐘的調控。我進一步於lwd1 lwd2 雙突變體中測試BPCs對CCA1啟動子的抑制。然而，由於在LWDs缺失時CCA1的表現量已大幅被抑制，無法看出BPCs對於CCA1有顯著的抑制效果。BPCs與LWDs的交互作用對於CCA1的調控仍有待近一步的研究。

Organisms on the earth keep their physiological processes including growth, development, and metabolism via receiving environmental day-night rhythms that entrain their internal timers, circadian clocks. In the model plant, Arabidopsis thaliana, the circadian clock system is constituted by the genes' regulatory feedback loops forming complicated networks. In recent years, it has been shown that LIGHT-REGULATED WD1 (LWD1) is a positive regulator of the clock dawn gene, CIRCADIAN CLOCK ASSOCIATED1 (CCA1). Mechanistically, LWD1 regulates clock genes via forming protein complexes with transcription factors that directly act on gene promoters. Our lab previously showed that LWD1 is transcriptionally co-expressed with family members of BASIC PENTACYSTEINEs (BPCs), the regulators crucial for the plant circadian growth. It has been reported that the absence of BPCs causes severe morphological phenotype and the overexpression of BPC3 could suppress the expression of CCA1. Even though the connection between LWDs and BPCs is observed, the biological significance of the co-expression between LWD1 and BPCs in circadian clock regulation is unknown. My research aim is to unravel the meaning of the LWD-BPC interaction. I examined if BPCs played a role in regulating clock dawn gene CCA1 and dusk gene GIGANTEA (GI) with molecular genetic analyses and found that the expressions of CCA1 and GI were reduced in bpc4 bpc6, bpc1 bpc2, and bpc1 bpc2 bpc3, indicating that BPCs are involved in the positive regulation of the circadian clock. I also proved that interaction between BPC1/BPC2/BPC6 and LWD1 by using bimolecular fluorescence complementation (BiFC), consistent with previous findings of the interaction between BPC3/4 and LWD1 in our lab. This suggested that BPCs might be involved in the circadian clock regulation with LWD1. I further examined if the interaction with LWD1 was required for the function of BPCs ectopic expression on CCA1 repression. The BPCs overexpression was introduced into the lwd1 lwd2 mutant. Nevertheless, since the expression of CCA1 was greatly reduced in the absence of LWDs, it cannot be seen if BPCs significantly inhibited the transcription of CCA1 in lwd1 lwd2 mutant. Therefore, further investigation is required for elucidating the role of BPC-LWD interaction in CCA1 regulation.