芳香烴碳氫化合物接受器抑制子(AHRR) 是屬於
basic-Helix-Loop-Helix (bHLH) Per-ARNT-Sim (PAS)家族蛋白的一
員。在老鼠中，AHRR 可被許多AHR 的ligand 所誘發，AHR 訊息傳
遞途徑可藉由戴奧辛(TCDD)及其他多環類化合物等之配體活化。當
AHR 訊息傳遞系統活化時，會啟動許多下游基因的轉錄表現，包括
cyp1a1 與Ahrr 等。AHRR 在哺乳動物中扮演AHR 訊息系統負迴饋
調控機制，藉由與AHR 競爭ARNT 的結合及XRE 序列的結合導致
AHR/ARNT 無法結合到目標基因調控區，進而抑制下游相關基因。
斑馬魚存在兩種AHRR，分別為AHRR1、AHRR2，各可轉譯出
550 和573 個胺基酸。由於斑馬魚ahrr1、ahrr2 上游訊息調控機制尚
不清楚，為了解斑馬魚中AHR 是否可以活化ahrr2，本實驗利用全覆
式原位雜交(whole-mount in situ hybridization)觀察ahrr2 的表現情況
以及弱化AHR 家族(ahr2、ahr1a 及ahr1b)後對ahrr2 基因表現的影
響。結果顯示 ahrr2 在1 細胞到12hpf 時期全身表現，而在24hpf 到
72hpf 時期主要表現在頭部。弱化ahr2、ahr1a 及ahr1b 在24hpf 時期
會些微抑制ahrr2 的表現，但在48hpf~96hpf 時對ahrr2 的抑制效果
不顯著。另外亦進行ahrr2 啟動子接合EGFP 報導基因質體的建構，
藉此觀察弱化AHR 家族(ahr2、ahr1a 及ahr1b)後ahrr2 啟動子的表
現情形。結果顯示無論在螢光強度或是發光比率上，任何AHR 家族
的弱化均不會影響ahrr2 啟動子的表現情形。分析ahrr2 啟動子發現，
序列中不存在XRE 序列，和哺乳類存在多個XRE 不同，推測可能在
斑馬魚中因為AHR 家族無法結合ahrr2 調節區域而不參與ahrr2 基
因的調控機制。

The aryl hydrocarbon receptor repressor (AHRR) is a transcription
factor belonging to the basic helix-loop-helix PAS (bHLH-PAS)
superfamily. The induction of AHRR by various AHR agonists was
investigated in mice and rats. In vertebrate, AHR mediates the biological
and toxic effects of various environmental pollutants, such as dioxin and
polycyclic aromatic compounds. Via AHR activation, a number of target
genes, including cyp1a1 and Ahrr, could be induced significantly. In
mammalian system, a negative regulatory loop of AHR-related signaling
pathway is established by AHR repressor (AHRR), which represses
AHR-related signaling pathway by competing with AHR for binding to
ARNT and xenobiotic response element(XRE).
In zebrafish, there are two forms of ahrr cDNAs have been cloned
that encode AHRR1 and AHRR2 with predicted size of 550 and 573
amino acids, respectively. The regulation of AHRR1 and AHRR2 still
remain to be studied. In this study, the regulation of ahrr2 has been
investigated by gene knockdown analysis. During developing stage,
ahrr2 is widely expressed through the whole embryo before 12 hpf.
Subsequently, it is mainly expressed in brain region until 72 hpf.
Blockage of individual ahr1a, ahr1b and ahr2 translation reduced ahrr2
transcription at 24 hpf but did not give significant effects after hatching.
A recombinant EGFP reporter vector, containing 1407 bp of ahrr2
upstream promoter sequence, was constructed. It appears that blockage of
individual ahr1a, ahr1b and ahr2 translation did not change the strength
and rate of transient EGFP expression. In addition, there is no putative xenobiotics response element in the upstream sequence of ahhr2 gene.
Taken together, zebrafish ahrr2 gene is different from the mammalian
ahrr gene and it is likely not regulated by AHR signaling pathway.

中文摘要································································································································ 1
英文摘要································································································································ 3
壹、 前言························································································································· 5
一、 芳香烴碳氫化合物接受器 (aryl hydrocarbon receptor，AHR)················5
二、 斑馬魚中AHR 及ARNT 蛋白在TCDD 發育毒性上所扮演的角色·····10
三、 AHR 和ARNT 蛋白在發育上扮演的角色·············································12
四、 芳香烴碳氫化合物接受器抑制蛋白(Aryl hydrocarbon receptor
repressor，AHRR)···················································································12
五、 研究動機·································································································17
貳、 實驗材料與方法·································································································· 19
參、 實驗結果··············································································································· 46
一、 斑馬魚ahrr2 基因在胚胎發育時期的表現············································46
二、 斑馬魚ahrr2 基因5’RACE ····································································46
三、 斑馬魚ahrr2 基因啟動子序列接合綠色螢光蛋白·································47
四、 斑馬魚ahrr2 基因上游序列啟動EGFP 報導基因螢光表現情形··········48
五、 pcDNA3.1-EGFP 質體DNA 螢光表現···················································49
六、 斑馬魚ahrr2 基因啟動子EGFP 全覆式原位雜交之表現·····················50
七、 斑馬魚ahrr2 基因調控與AHR 家族訊息傳遞系統之關係···················50
肆、 討論······················································································································· 54
一、 斑馬魚ahrr2 基因在胚胎發育時期的表現············································54
二、 斑馬魚ahrr2 基因調控與AHR 家族訊息傳遞系統之關係···················55
三、 zfAHRR2 啟動子1.4K-EGFP 表現之探討·············································56
伍、 實驗結果-表········································································································· 58
陸、 實驗結果-圖········································································································· 64
一、 建構AHRR2 promoter(1.4K)-EGFP 流程··············································64
二、 斑馬魚胚胎10hpf 之前ahrr2 基因之表現·············································65
三、 斑馬魚胚胎時期ahrr2 基因之表現(續) ·················································66
四、 斑馬魚ahrr2 基因5’RACE ····································································67
五、 ahrr2 轉譯起始點上游序列1407bp 的選殖············································68
六、 斑馬魚ahrr2 基因啟動子序列接合綠色螢光蛋白································69
七、 斑馬魚ahrr2 基因啟動子序列EGFP(50pg)螢光表現···························69
八、 斑馬魚ahrr2 基因啟動子序列EGFP(100pg)螢光表現··························70
九、 斑馬魚ahrr2 基因啟動子序列EGFP(150pg)螢光表現··························72
十、 顯微注射pcDNA3.1-EGFP vector ··························································73
十一、注射AHRR2 1.4K-EGFP 後斑馬魚胚胎時期EGFP 之表現·················74
十二、注射AHR1B-MO 12ng 後AHR1B-EGFP 在24hpf 時期的表現···········75
十三、注射AHR2、AHR1A 及AHR1B –MO 後斑馬魚胚胎24hpf 時期ahrr2
基因之表現·····························································································76
十四、注射AHR2、AHR1A 及AHR1B –MO 後斑馬魚胚胎48hpf 時期ahrr2
基因之表現·····························································································77
十五、注射AHR2、AHR1A 及AHR1B –MO 後斑馬魚胚胎72hpf 時期ahrr2
基因之表現·····························································································78
十六、注射AHR2、AHR1A 及AHR1B –MO 後斑馬魚胚胎96hpf 時期ahrr2
基因之表現·····························································································79
十七、顯微注射AHR1A-MO (12ng) + pAHRR2 1.4-EGFP (50pg) ··················80
十八、顯微注射AHR1B –MO(12ng) +AHRR2 1.4K-EGFP (50pg) ··················81
十九、顯微注射AHR2-MO (12ng) + pAHRR2 1.4-EGFP (50pg) ·····················82
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