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研究生(外文):Chun-Hung Lu
論文名稱(外文):Characterization of the Regulatory Elements of the Zebrafish Type II Keratin Gene, zfCKII
指導教授(外文):Huai-Jen Tsai
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中間絲蛋白(intermediate filament)共分成五大類,第一型角質蛋白(type I keratin)及第二型角質蛋白(type II keratin)分屬其中最主要的兩大類別。在哺乳類動物之中,角質蛋白主要是表現在上皮細胞,並且已知至少有30個基因可以表現出角質蛋白,調控機制和作用的調控因子也有一些研究。然而,在斑馬魚(Danio rerio)中,雖然對於角質蛋白如表現位置和時間等特性已經有了很多的研究,但是目前角質蛋白基因在胚胎發育時的調控機制尚不清楚。在本文中,我們選殖出一段斑馬魚第二型角質蛋白基因(zebrafish type II cytokeratin, zfCKII)的上游調控區-2274到-1(-2274/-1)的序列,利用不同長度上游序列來驅動紅螢光蛋白基因,透過基因轉殖技術觀察其在轉殖魚中各組織的表現狀態。結果顯示,zfCKII上游DNA片段含-2274/-1、-1500/-1、-1000/-1、-600/-1、-500/-1、-400/-1、和-200/-1皆能驅使紅螢光報導基因在皮膚表現紅螢光。在-100/-1和-90/-1中則完全無紅螢光表現。因此,綜合這些結果顯示-200/-1片段是驅動紅螢光報導基因在皮膚表現的最小DNA調控片段。然而,在-1500/-1、-1000/-1、-600/-1、和-500/-1除了皮膚外,分別各有52、64、49及68%在肌肉部位表現出紅螢光,顯示-2274/-1500可能具有抑制肌肉表現的調控區存在,雖然將此段序列接到胚胎早期肌肉細胞命運決定因子myf-5基因-300/-1的上游來驅動綠螢光蛋白基因,並無明顯的抑制作用。進一步地,分析斑馬魚-200/-100序列,發現-151/-121包含可能和轉錄因子krüppel-like factor結合的片段。若將-146/143上原本的GGGG突變為AAAA,會使得DNA調控片段幾乎失去驅動紅螢光報導基因在皮膚上表現的能力。故此(-146/143)片段對於斑馬魚角質蛋白基因在皮膚中的表現是十分重要的。
Type I (acidic) and type II (basic) keratins, two major classes of intermediate filament proteins, is encoded by at least 30 genes and are mainly expressed in epithelial cells in mammals. The regulation of keratin expression and transcription factors have been studied in mammals. However, the role of regulation is still unclear although there are many studies on the expression pattern of keratin in zebrafish (Danio rerio). In this report, we characterized an upstream 2274 bp (-2274/-1) of zebrafish type II cytokeratin (zfCKII) by transgenic analysis. Series of upstream deletions linked to the red fluorescent protein (RFP) reporter were constructed and microinjected into one-cell fertilized eggs. Transient transgenic analysis showed that the segments of -2274/-1, -1500/-1, -1000/-1, -600/-1, -500/-1, -400/-1 and -200/-1 enabled to direct RFP to be expressed in skin. But, the expression of RFP was completely abolished in the transgenic fish when the segment of -90/-1 and -100/-1 were transferred, indicating that -200/-1 is the minimal promoter to drive RFP expression in skin. Nevertheless, the segments of -1500/-1, -1000/-1, -600/-1, and -500/-1 were capable of directing RFP expression not only in skin but also in skeletal muscle. The expression rate in muscle ranged from 50 to 70%, compared to 0% of -2274/-1-injected embryos. Therefore, we propose that a muscle repressive element may exist in -2274/-1500, although the GFP expression rate in muscle of embryos injected with -2274/-1500 fused with -300/-1 of muscle regulatory factor myf-5 did not decrease significantly. Sequence analysis of zfCKII -200/-100 showed that there is a putative krüppel-like factor binding site located at -151/-121. When we mutated the segment -146/-143 from GGGG to AAAA, we found that this mutated segment almost lost its function to drive RFP expression in skin, suggesting that -146/-143 is an essential sequence for keratin expression in the skin of zebrafish.
中文摘要 1
Abstract 3
壹、 前言 4
貳、 實驗材料 10
參、 實驗方法 12
肆、 結果 21
伍、 討論 26
陸、 參考資料 32
圖表 36
附錄(一) 斑馬魚胚胎發育時期圖 45
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