(3.226.72.118) 您好!臺灣時間:2021/05/13 06:54
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:陳美均
研究生(外文):Mei-Chun Chen
論文名稱:斑馬魚Arylhydrocarbonreceptorrepressor2(Ahrr2)啟動子的分析
論文名稱(外文):The characterization of the Aryl hydrocarbon receptor repressor 2(Ahrr2) promoter from the zebrafish.
指導教授:胡清華胡清華引用關係
指導教授(外文):Chin-Hwa Hu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:95
中文關鍵詞:斑馬魚芳香烴碳氫化合物接受器芳香烴碳氫化合物接受器抑制子
外文關鍵詞:zebrafishAHRAHRR
相關次數:
  • 被引用被引用:0
  • 點閱點閱:178
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
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
柒、 參考文獻··············································································································· 83
捌、 附錄······················································································································· 89
Abnet, C. C., Tanguay, R. L., Hahn, M. E., Heideman, W. and
Peterson, R. E. (1999). Two forms of aryl hydrocarbon receptor type 2 in
rainbow trout (Oncorhynchus mykiss). Evidence for differential
expression and enhancer specificity. J Biol Chem 274, 15159-15166.
Andreasen, E. A., Hahn, M. E., Heideman, W., Peterson, R. E. and
Tanguay, R. L. (2002). The zebrafish (Danio rerio) aryl hydrocarbon
receptor type 1 is a novel vertebrate receptor. Mol Pharmacol 62,
234-249.
Baba, T., Mimura, J., Gradin, K., Kuroiwa, A., Watanabe, T.,
Matsuda, Y., Inazawa, J., Sogawa, K. and Fujii-Kuriyama, Y. (2001).
Structure and expression of the Ah receptor repressor gene. J Biol Chem
276, 33101-33110.
Bernshausen, T., Jux, B., Esser, C., Abel, J. and Fritsche, E. (2006).
Tissue distribution and function of the Aryl hydrocarbon receptor
repressor (AhRR) in C57BL/6 and Aryl hydrocarbon receptor deficient
mice. Arch Toxicol 80, 206-211.
Carney, S. A., Peterson, R. E. and Heideman, W. (2004).
2,3,7,8-Tetrachlorodibenzo-p-dioxin activation of the aryl hydrocarbon
receptor/aryl hydrocarbon receptor nuclear translocator pathway causes
developmental toxicity through a CYP1A-independent mechanism in
zebrafish. Mol Pharmacol 66, 512-521.
Carney, S. A., Prasch, A. L., Heideman, W. and Peterson, R. E. (2006).
Understanding dioxin developmental toxicity using the zebrafish model.
Birth Defects Res A Clin Mol Teratol 76, 7-18.
Cauchi, S., Stucker, I., Cenee, S., Kremers, P., Beaune, P. and
Massaad-Massade, L. (2003). Structure and polymorphisms of human
aryl hydrocarbon receptor repressor (AhRR) gene in a French population:
relationship with CYP1A1 inducibility and lung cancer.
Pharmacogenetics 13, 339-347.
Coumailleau, P., Poellinger, L., Gustafsson, J. A. and Whitelaw, M. L.
(1995). Definition of a minimal domain of the dioxin receptor that is
associated with Hsp90 and maintains wild type ligand binding affinity
and specificity. J Biol Chem 270, 25291-25300.
Davarinos, N. A. and Pollenz, R. S. (1999). Aryl hydrocarbon receptor
imported into the nucleus following ligand binding is rapidly degraded
via the cytosplasmic proteasome following nuclear export. J Biol Chem
274, 28708-28715.
Evans, B. R., Karchner, S. I., Allan, L. L., Pollenz, R. S., Tanguay, R.
L., Jenny, M. J., Sherr, D. H. and Hahn, M. E. (2007). Repression of
aryl hydrocarbon receptor (AHR) signaling by AHR repressor (AHRR):
Role of DNA binding and competition for ARNT. Mol Pharmacol.
Evans, B. R., Karchner, S. I., Franks, D. G. and Hahn, M. E. (2005).
Duplicate aryl hydrocarbon receptor repressor genes (ahrr1 and ahrr2) in
the zebrafish Danio rerio: structure, function, evolution, and
AHR-dependent regulation in vivo. Arch Biochem Biophys 441, 151-167.
Fujita, H., Kosaki, R., Yoshihashi, H., Ogata, T., Tomita, M.,
Hasegawa, T., Takahashi, T., Matsuo, N. and Kosaki, K. (2002).
Characterization of the aryl hydrocarbon receptor repressor gene and
association of its Pro185Ala polymorphism with micropenis. Teratology
65, 10-18.
Ge, N. L. and Elferink, C. J. (1998). A direct interaction between the
aryl hydrocarbon receptor and retinoblastoma protein. Linking dioxin
signaling to the cell cycle. J Biol Chem 273, 22708-22713.
Gu, Y. Z., Hogenesch, J. B. and Bradfield, C. A. (2000). The PAS
superfamily: sensors of environmental and developmental signals. Annu
Rev Pharmacol Toxicol 40, 519-561.
Haarmann-Stemmann, T. and Abel, J. (2006). The arylhydrocarbon
receptor repressor (AhRR): structure, expression, and function. Biol
Chem 387, 1195-1199.
Hahn, M. E., Karchner, S. I., Shapiro, M. A. and Perera, S. A. (1997).
Molecular evolution of two vertebrate aryl hydrocarbon (dioxin)
receptors (AHR1 and AHR2) and the PAS family. Proc Natl Acad Sci U S
A 94, 13743-13748.
Hill, A. J., Teraoka, H., Heideman, W. and Peterson, R. E. (2005).
Zebrafish as a model vertebrate for investigating chemical toxicity.
Toxicol Sci 86, 6-19.
Ikuta, T., Eguchi, H., Tachibana, T., Yoneda, Y. and Kawajiri, K.
(1998). Nuclear localization and export signals of the human aryl
hydrocarbon receptor. J Biol Chem 273, 2895-2904.
Karchner, S. I., Franks, D. G. and Hahn, M. E. (2005). AHR1B, a new
functional aryl hydrocarbon receptor in zebrafish: tandem arrangement of
ahr1b and ahr2 genes. Biochem J 392, 153-161.
Karchner, S. I., Franks, D. G., Powell, W. H. and Hahn, M. E. (2002).
Regulatory interactions among three members of the vertebrate aryl
hydrocarbon receptor family: AHR repressor, AHR1, and AHR2. J Biol
Chem 277, 6949-6959.
Kawajiri, K. and Fujii-Kuriyama, Y. (2007). Cytochrome P450 gene
regulation and physiological functions mediated by the aryl hydrocarbon
receptor. Arch Biochem Biophys 464, 207-212.
Kikuchi, Y., Ohsawa, S., Mimura, J., Ema, M., Takasaki, C., Sogawa,
K. and Fujii-Kuriyama, Y. (2003). Heterodimers of bHLH-PAS protein
fragments derived from AhR, AhRR, and Arnt prepared by co-expression
in Escherichia coli: characterization of their DNA binding activity and
preparation of a DNA complex. J Biochem 134, 83-90.
Korkalainen, M., Linden, J., Tuomisto, J. and Pohjanvirta, R. (2005).
Effect of TCDD on mRNA expression of genes encoding bHLH/PAS
proteins in rat hypothalamus. Toxicology 208, 1-11.
Korkalainen, M., Tuomisto, J. and Pohjanvirta, R. (2004). Primary
structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a
TCDD-resistant rat strain. Biochem Biophys Res Commun 315, 123-131.
Lees, M. J. and Whitelaw, M. L. (1999). Multiple roles of ligand in
transforming the dioxin receptor to an active basic helix-loop-helix/PAS
transcription factor complex with the nuclear protein Arnt. Mol Cell Biol
19, 5811-5822.
McMillan, B. J. and Bradfield, C. A. (2007). The aryl hydrocarbon
receptor sans xenobiotics: endogenous function in genetic model systems.
Mol Pharmacol 72, 487-498.
Mimura, J., Ema, M., Sogawa, K. and Fujii-Kuriyama, Y. (1999).
Identification of a novel mechanism of regulation of Ah (dioxin) receptor
function. Genes Dev 13, 20-25.
Mimura, J. and Fujii-Kuriyama, Y. (2003). Functional role of AhR in
the expression of toxic effects by TCDD. Biochim Biophys Acta 1619,
263-268.
Nishihashi, H., Kanno, Y., Tomuro, K., Nakahama, T. and Inouye, Y.
(2006). Primary structure and organ-specific expression of the rat aryl
hydrocarbon receptor repressor gene. Biol Pharm Bull 29, 640-647.
Prasch, A. L., Heideman, W. and Peterson, R. E. (2004). ARNT2 is not
required for TCDD developmental toxicity in zebrafish. Toxicol Sci 82,
250-258.
Prasch, A. L., Tanguay, R. L., Mehta, V., Heideman, W. and Peterson,
R. E. (2006). Identification of zebrafish ARNT1 homologs:
2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish
requires ARNT1. Mol Pharmacol 69, 776-787.
Prasch, A. L., Teraoka, H., Carney, S. A., Dong, W., Hiraga, T.,
Stegeman, J. J., Heideman, W. and Peterson, R. E. (2003). Aryl
hydrocarbon receptor 2 mediates 2,3,7,8-tetrachlorodibenzo-p-dioxin
developmental toxicity in zebrafish. Toxicol Sci 76, 138-150.
Roy, N. K., Courtenay, S. C., Chambers, R. C. and Wirgin, II. (2006).
Characterization of the aryl hydrocarbon receptor repressor and a
comparison of its expression in Atlantic tomcod from resistant and
sensitive populations. Environ Toxicol Chem 25, 560-571.
Sogawa, K., Iwabuchi, K., Abe, H. and Fujii-Kuriyama, Y. (1995).
Transcriptional activation domains of the Ah receptor and Ah receptor
nuclear translocator. J Cancer Res Clin Oncol 121, 612-620.
Tanguay, R. L., Abnet, C. C., Heideman, W. and Peterson, R. E.
(1999). Cloning and characterization of the zebrafish (Danio rerio) aryl
hydrocarbon receptor. Biochim Biophys Acta 1444, 35-48.
Tanguay, R. L., Andreasen, E., Heideman, W. and Peterson, R. E.
(2000). Identification and expression of alternatively spliced aryl
hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with
distinct functions. Biochim Biophys Acta 1494, 117-128.
Taylor, B. L. and Zhulin, I. B. (1999). PAS domains: internal sensors of
oxygen, redox potential, and light. Microbiol Mol Biol Rev 63, 479-506.
Tsuchiya, Y., Nakajima, M., Itoh, S., Iwanari, M. and Yokoi, T. (2003).
Expression of aryl hydrocarbon receptor repressor in normal human
tissues and inducibility by polycyclic aromatic hydrocarbons in human
tumor-derived cell lines. Toxicol Sci 72, 253-259.
Watanabe, T., Imoto, I., Kosugi, Y., Fukuda, Y., Mimura, J., Fujii, Y.,
Isaka, K., Takayama, M., Sato, A. and Inazawa, J. (2001). Human
arylhydrocarbon receptor repressor (AHRR) gene: genomic structure and
analysis of polymorphism in endometriosis. J Hum Genet 46, 342-346.
Wentworth, J. N., Buzzeo, R. and Pollenz, R. S. (2004). Functional
characterization of aryl hydrocarbon receptor (zfAHR2) localization and
degradation in zebrafish (Danio rerio). Biochem Pharmacol 67,
1363-1372.
Yamamoto, J., Ihara, K., Nakayama, H., Hikino, S., Satoh, K., Kubo,
N., Iida, T., Fujii, Y. and Hara, T. (2004). Characteristic expression of
aryl hydrocarbon receptor repressor gene in human tissues: organ-specific
distribution and variable induction patterns in mononuclear cells. Life Sci
74, 1039-1049.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔