BPC家族於阿拉伯芥生理時鐘調節之遺傳分析

Abstract

爲因應地球自轉產生的晝夜輪替，生物體內的生理時鐘(circadian clock)能整合外在週期性的環境變化與內在基因調控，進而調節各種生理途徑成規律的變化。目前已知阿拉伯芥(Arabidopsis thaliana)的生理時鐘是由許多基因調控迴圈組成的基因網絡。先前研究指出LIGHT-REGULATED WD1 (LWD1) 能正向調控阿拉伯芥生理時鐘，此外也發現植物特有的轉錄因子BASIC PENTACYSTEINE (BPC)家族與LWD1晝夜節律的表現模式類似，且BPC突變株具有多重生理時鐘調控的外顯缺陷，因此本研究進一步探討BPC家族於阿拉伯芥生理時鐘的調節所扮演的角色，本論文進行三種不同遺傳性分析策略來測定BPC家族對於阿拉伯芥生理時鐘的調控：(一) 建立可抑制BPC基因表現的artificial mircoRNA-BPCs（amiR-BPCs）誘導構築，並檢測抑制BPC表現時植物生理時鐘基因表現是否被影響; (二) 利用小鼠的Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (mAPOBEC1) 去胺基酶融合於BPC3/4蛋白，檢測BPC3/4 可能結合的時鐘基因啟動子; (三) 利用雙分子螢光互補作用(bimolecular fluorescence complementation, BiFC) 檢測LWD1及BPC3/4蛋白質之間的交互作用。本研究於帶有時鐘基因報導子之阿拉伯芥中分別轉殖amiR-BPC1/2以及BPC3/4-mAPOBEC1構築。目前測試轉植株分別在amiR-BPC1/2誘導前後對於pCCA1及pGI報導子皆無明顯改變，但仍須進一步檢測amiRNA是否能有效抑制BPC表現。測試mAPOBEC1與BPC3/4融合蛋白之轉殖株時發現mAPOBEC1融合可能使BPC3/4失去原先的功能，並不適用作為研究BPC功能之工具。在LWD1與BPC3/4 BiFC交互作用測試中，我已成功偵測到 LWD1與BPC3和BPC4在細胞中的交互作用，顯示LWD1可與BPC3或BPC4在細胞中確實可形成蛋白質複合體。

To fit external changes upon the day-night cycle, the internal circadian clock of organism adjusts various physiological pathways according to the external rhythmic stimuli. The circadian clock of Arabidopsis thaliana is a complex network composing of feedback regulatory gene loops. Previous studies have shown that LIGHT-REGULATED WD1 (LWD1), one of clock components, positively regulates the promoters of clock genes without a putative DNA-binding domain. In a LWD1 coexpression assay, the transcriptional profiles of BASIC PENTACYSTEINE (BPC) members were coexpressed with that of the LWD1 under the day-night cycle. It has been reported that the high order mutant of BPCs shows multiple defects in clock-regulated phenotypes. We therefore investigated the role of BPC family in the regulation of the circadian clock. We construct an inducible amiRNA targeting the BPC members to examine whether the clock behavior would be affected while the repression of BPCs was induced. Furthermore, we fused BPC3 and BPC4 respectively with the mouse apolipoprotein B mRNA editing enzyme catalytic polypeptide 1 (mAPOBEC1), a deaminase, for examining BPC3/4 in vivo targeting sites of promoters of clock genes. We also tested the interaction between LWD1 and BPC3/4 by conducting bimolecular fluorescence complementation (BiFC) in Arabidopsis seedlings. We are currently testing the functions of amiRNA and BPC3/4-mAPOBEC1 constructs in the transgenic lines carrying the clock reporter. In the preliminary data, neither the profile of pCCA1 nor that of pGI reporter was changed under the induction of amiR-BPC1/2. We would further examine if BPC expressions were inhibited by the amiR-BPC1/2. By analyzing transgenic plants carrying mAPOBEC1 fused BPC3/4 proteins, we found that mAPOBEC1 fusion may have impeded the function of BPC3/4 protein and not suitable for the study. BiFC signals were successfully detected by coexpressing LWD1 with BPC3 or BPC4 in Arabidopsis seedlings, indicating that LWD1 would form with BPC3 or BPC4 a protein complex in cells.