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研究生:陳宏彥
研究生(外文):Hung-Yen Chen
論文名稱:斑馬魚Agr2基因表現的調控機制之研究
論文名稱(外文):Study of regulatory mechanisms of anterior gradient 2 (Agr2) gene expression in zebrafish
指導教授:黃聲蘋
指導教授(外文):Sheng-Ping L.Hwang
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:102
中文關鍵詞:內胚層發育前端梯度2杯狀細胞
外文關鍵詞:endodermdevelopmentAgr2goblet cellintestinepharynx
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本論文的主要目的是要研究Agr2基因表現的調節機制,首先利用Ensembl Genome Browser搜尋引擎搜尋斑馬魚Agr2 upstream /promoter DNA序列與轉錄區之DNA、使用promoter scan分析Agr2預測保守的promoter region及TATA Box位置、利用Family Relations II軟體比較不同物種之Agr2 upstream/promoter DNA 序列來預測是否有保守的區域及利用網站Genomatix中的Matinspector軟體來預測了6kbp Agr2 upstream/promoter DNA序列可能有哪些轉錄因子結合區域,上述所獲得之資訊用來設計製備不同長度的Agr2 upstream /promoter DNA sequence接合入TolⅡ-EGFP transposon vector中,分別獲得:[-6038EGFP]、[-5276EGFP]、[-4188EGFP]、[-3284EGFP]與[-2049EGFP]質體DNA與transposase mRNA共同注射入斑馬魚胚胎1~2細胞時期,觀察從24~120 hpf間綠色螢光蛋白表現位置,結果顯示[-6038EGFP]和[-5276EGFP]注射的斑馬魚胚胎觀察到的綠色螢光蛋白表現位置與內生性Agr2 mRNA表現位置相似度很高,觀察結果顯示24~120hpf期間綠色螢光蛋白主要會表現在孵化腺、黏液細胞、耳半規管、咽、食道、腸道杯狀細胞;[-4188EGFP]與[-3284EGFP]注射的斑馬魚胚胎所觀察到的綠色螢光蛋白表現位置只在耳半規管與黏液細胞,而其內胚層衍生器官如咽與腸道杯狀細胞則無綠色螢光蛋白表現;最後[-2049EGFP]注射的斑馬魚胚胎多數胚胎則無綠色螢光蛋白表現,若有綠色螢光蛋白表現則為非專一性的在肌肉或脊索的mosaic pattern。綜而言之,Agr2 upstream /promoter DNA序列在-5276至-4188有調控綠色螢光蛋白表現在不同內胚層衍生器官如咽與腸道杯狀細胞之功能,而在 -3284至-2049間則具有調控綠色螢光蛋白表現在耳半規管與黏液細胞的調節序列。最後將[-6038EGFP]注射的斑馬魚胚胎所觀察綠色螢光蛋白表現與內生性Agr2 mRNA表現位置相仿的斑馬魚胚胎經過三個月的飼養,經交配所生下之F1子代,觀察發現F1子代綠色螢光蛋白表現位置與內生性Agr2 mRNA分佈情形可說是幾乎完全相符。
The main goal of my thesis is to study the regulatory mechanism of anterior gradient 2 (Agr2) gene expression in zebrafish. At the begining, I used Ensembl Genome Browser to search and download zebrafish Agr2 upstream/promoter DNA sequence, used Promoter scan to predict potential promoter and TATA box regions, used Family Relations II to search for conserved regions of Agr2 upstream/promoter DNA among different species, and used Matinspector of Genomatix website to localize possible transcription factor binding motifs in 6kbp Agr2 upstream/ promoter DNA sequence. The obtained information was used to design different lengths of Agr2 upstream/promoter DNA sequences in order to ligate them into Tol II-EGFP transposon vector. Thus, I have obtained respective recombinant plasmids including [-6038EGFP], [-5276EGFP], [-4188EGFP], [-3284EGFP] and [-2049EGFP] plasmids. After Co-injection of both the respective plasmid and transposase mRNA into 1~2 cells of zebrafish embryo, EGFP expression pattern in injected embryos was monitored during 24 to 120 h of development. Results of EGFP expression pattern of respective [-6038EGFP]- and [-5276EGFP]-injected embryos show that they share high similarity with endogenous Agr2 mRNA expression pattern. EGFP is mainly expressed in hatching gland, mucous cells, otic vesicle, pharynx, esophagus, and goblet cells in the intestine. EGFP expression patterns in respective [-4188EGFP]- and [-3284EGFP]-injected embryos are restricted in mucous cells and otic vesicles, while no EGFP expression can be detected in any endoderm derived organs such as pharynx and goblet cells in the intestine. Most of [-2049EGFP]-injected embryos show either no EGFP expression or non-specific muscle or notochord expression. In conclusion, DNA sequence within -5276~ -4188 of Agr2 upstream/promoter may regulate EGFP expression in endoderm derived organs such as pharynx and goblet cells in the intestine, while DNA sequence within -3284~ -2049 of Agr2 upstream/promoter may regulate EGFP expression in otic vesicles and mucous cells. Finally, those [-6038EGFP]-injected embryos showing EGFP expression pattern similar to endogenous Agr2 mRNA localization were raised to adulthood after three months. EGFP expression patterns in F1 embryos from Tg(Agr2 6K:EGFP) transgenic fish is the same as endogenous Agr2 mRNA distribution during 24 to 120
hpf of development.
誌謝 i
中文摘要 iii
英文摘要 v
目錄 vii
實驗方法目錄 viii
圖目錄 x
前言 1
實驗方法 21
實驗結果 43
討論 49
參考文獻 56
圖表 62
附錄 86
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