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研究生:蔡青彥
研究生(外文):Chin-Yan Tsai
論文名稱:斑馬魚低氧誘發蛋白hifα轉錄因子對於斑馬魚vsx1基因上游啟動子之基因調控作用
論文名稱(外文):Functions of hypoxia-inducible factors (HIFs) in vsx1 transcriptions during zebrafish developmental stage
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:2009
畢業學年度:97
語文別:中文
論文頁數:71
中文關鍵詞:低氧誘發蛋白
外文關鍵詞:HIF-avsx1
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bHLH-PAS蛋白在脊椎動物的胚胎發育過程中扮演著重要的角色,其中低氧誘發蛋白HIF (Hypoxia induce factors)主要的功能在於調節生物體內的供氧平衡(oxygen homeostasis),在細胞遇到缺氧的情況下,會與ARNT結合而形成異偶合體結構(heterodimer),進而影響到下游基因表現以調控細胞缺氧時的反應。
近來人類有很多視網膜方面的疾病,被指出與HIFα蛋白有密切的關係。此外,視網膜發育的表現基因:vsx1(visual system homeobox 1)產生突變,也會造成視網膜兩極細胞無法分化造成彩色視覺受到影響。
先前的研究發現,在斑馬魚hif-1α/2α/3α mix morpholino基因弱化(knockdown)實驗中,會造成視網膜發育中的兩極細胞(bipolar cell)發育受到影響,其中主要標示基因:Vsx1表現量與表現範圍明顯受到抑制。因此本實驗中主要分析斑馬魚Vsx1基因上游啟動子序列,發現含有多個HIF-α/ARNT偶合體辨識並結合的位置:HREs (Hypoxia response elements)。本實驗中以聚合?連鎖反應夾取包含HREs的不同長度片段,並接入螢光報導載體: T2KXIGDin中,以顯微注射至ㄧ細胞胚胎內觀察螢光表現的強度以及專一性。另外注射Hif-1α、2α、3α morpholino個別弱化其標的基因以觀察內生性的vsx1基因表現,以及含有vsx1啟動子的螢光報導基因表現為何。結果發現,Hif-2α可能透過辨識vsx1上游的HRE序列來調控vsx1基因的表現。這項實驗首次找出負責調控兩極細胞vsx1基因的低氧誘發因子,進而確認低氧誘發訊息傳遞系統在視網膜細胞分化中所扮演的角色。
bHLH-PAS is a protein family that play important functions in vertebrate development, cellular homeostasis, and metabolic balance. It includes the aryl hydrocarbon receptor nuclear translocator (ARNT) and its associate factor, hypoxia-inducible factor alpha subunit (HIF-α). The unstable HIF-α subunit act as intracellular oxygen sensor and it is stabilized by oxygen deficiency. Under hypoxia stress treatment, HIF-αsubunit associates with ARNT to form a heterodimeric complex and binds specifically to a 5’-RCGTG-3’ hypoxia-responsive element (HRE) in the promoter or enhancer of various hypoxia inducible genes to stimulate transcription of target genes.
Previously, it was revealed that blocking hif-1α/2α/3α translation caused small eye, incomplete retina development and defect bipolar and rod cell differentiation. Retinal markers vsx1 were all repressed in hif-1α/2α/3α morphant. In the upstream region of vsx1 contains several hypoxia response elements (HRE). It is speculated that vsx1 is probably requlated by HIFs.
To examine the role of HIF on the transcriptional activity of vsx1 upstream sequence, I have fused the upstream sequence of vsx1 with the codon sequence of enhanced green fluorescence protein (EGFP).The expression efficiency of reporter gene was examined individually in each hif-α knockdown morphant embryo. It appears that HIF-2α konckdown supressed the reporter expression and suggests that HIF-2α has important roles in vsx1 transcription and retina differentiation
摘要 i
Abstract iii
目錄 iv
壹.前言 1
ㄧ.低氧誘發蛋白(Hypoxia- induce-factor) Hifα簡介 1
二.細胞內氧濃度與低氧誘發蛋白結構穩定性 2
三.斑馬魚眼睛結構和發育過程: 3
四.Vsx1簡介: 6
五.Tol2 載體 system : 7
A.跳躍子 7
B.Tol2簡介 7
六.實驗動機與目的 9
貳.實驗方法: 10
一、斑馬魚vsx1基因5’端上游啟動子之選殖 10
A. Zebrafish Genomic DNA (total DNA) 萃取 10
B. 斑馬魚vsx1基因5’端上游啟動子搜尋 11
C. 斑馬魚vsx1基因5’ 端上游啟動子DNA片段之選殖 13
D. 聚合?連鎖反應 (polymerase chain reaction , PCR) 13
E. DNA洋菜膠電泳 14
F. 聚合?連鎖反應產物之純化 14
G.重組DNA接合反應 (Ligation) 15
H. 質體轉型 (Trsnsformation) 15
I. DNA分子之萃取 (DNA Extraction) 16
J. 選殖質體DNA之正確性判斷 18
K. 啟動子與綠色螢光蛋白載體(Tol2-EGFP vector)之構築 19
L. 單點突變實驗(site direct mutation) 20
二、 斑馬魚胚胎顯微注射 (Microinjection) 22
A. 斑馬魚之飼養 22
B. 班馬魚受精卵收集 22
C. 顯微注射針(Micropipette)的準備 23
D. 洋菜膠平台(蛋盤)的製備 23
E. 專一性寡核酸 ( antisense morpholino,MO)設計 24
F. 配製實驗注射hif-1α/hif-2α/ hif-3α MO之溶液 24
G. 配製實驗注射綠色光蛋白載體之溶液 24
H. 顯微注射 24
I. 螢光顯微鏡觀察及拍照 25
J. RNA全覆式原位雜交 (RNA whole-mount in situ hybridization) 25
?. 實驗結果 32
一. 弱化Hif-α基因對胚胎內生性vsx1表現的影響 32
二. vsx1上游啟動子序列分析 33
三. Tg (Vsx1:EGFP)轉基因魚報導基因的表現 34
四.弱化Hif-α基因對vsx1-3.5K-EGFP螢光表現的影響 35
五.單點突變3.5K上pax6序列螢光表現 35
肆.討論 37
伍. 圖片 42
圖1. 弱化Hif-α基因對內生性vsx1基因的影響: 42
圖2. vsx1啟動子報導質體在胚胎內表現情形: 43
圖3. 弱化Hif-α基因 對pVsx1-3.5K-EGFP質體報導基因的影響: 44
圖4.-3.2K上Pax6序列突變對vsx1啟動子的影響 45
圖5.斑馬魚vsx1基因5’端上游啟動子5000base pair序列: 48
圖6. Vsx1上游-4986bp序列低氧反應原件(HRE)分析及PCR反應引子設計: 49
圖7.vsx1上游啟動子上Pax6轉錄因子結合位置以及PCR引子設計 50
陸.附圖 51
附圖1.BHLH-PAS蛋白結構示意 51
附圖2.HIF-α蛋白結構相似性比較 51
附圖3. T2KXIGDin載體結構圖及限制?切位 52
附圖4.人類及老鼠vsx1上游-5000bp序列低氧反應原件(HRE)分析 53
柒.參考文獻 54
捌.實驗材料 58
朱國榮。2007。bHLH/PAS蛋白質在斑馬魚胚胎的眼睛發育過程中所扮演的角色。碩士論文。國立台灣海洋大學生物科技研究所。

劉宗麟。 2008。斑馬魚低氧誘發蛋白HIFα對於斑馬魚眼睛發育中rho與vsx1基因上游啟動子之基因調控。碩士論文。國立台灣海洋大學生物科技研究所。

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