系統識別號 U0026-0812200913593718
論文名稱(中文) 斑馬魚10-甲醛四氫葉酸去氫酶啟動子區域的選殖與特性研究
論文名稱(英文) Cloning and characterization of the zebrafish 10-formyltetrahydrofolate dehydrogenase promoter region
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 95
學期 2
出版年 96
研究生(中文) 鍾宜孝
研究生(英文) I-hsiao Chung
學號 t3694103
學位類別 碩士
語文別 中文
論文頁數 97頁
口試委員 指導教授-傅子芳
中文關鍵字 斑馬魚 
英文關鍵字 zebrafish 
中文摘要 10-甲醛四氫葉酸去氫酶(10-formyltetrahydrofolate dehydrogenase, FDH)可以將10-甲醛四氫葉酸(10-formyltetrahydrofolate, 10-formyl-THF)轉換成四氫葉酸(tetrahydrofolate, THF)。過去研究發現在大量增生的癌細胞株中FDH的表現會受到抑制。而過量的表現FDH則會造成細胞週期阻斷以及細胞程式化凋亡。因此FDH的表現及調控被認為可能與腫瘤生成和胚胎發育有關。在本研究中我們藉由核酸選殖技術將斑馬魚10-甲醛四氫葉酸去氫酶(zFDH)的啟動子區域 (promoter region) 約1703 bp之片段,從斑馬魚染色體放大並選殖入pGL-3載體,以決定啟動子活性。並從斑馬魚5’-RACE cDNA library中選殖出FDH之5’不轉譯區(5’UTR) 以決定轉錄的起始點。在經過連續刪除縮減promoter並測試其活性後,發現-124到+40的區域為具promoter活性之最小片段。同時+1到+40的exon1區域對promoter活性是必需的。利用-124/+40片段為探針進行EMSA發現有特異性的蛋白質與核苷酸的複合物產成。針對此164 bp片段做序列分析後,發現有Sp1及c-Myb的結合位。單點突變(Site-directed mutagenesis)將Sp1結合位置突變後造成promoter活性有意義的下降。但c-Myb結合位的突變則無明顯的變化。我們同時將斑馬魚的Sp1蛋白的編碼序列(coding sequence)選殖到表現蛋白的載體pET43.1a及pcDNA3.1 myc/his中,表現斑馬魚Sp1蛋白並且純化。
英文摘要 10-formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyl- tetrahydrofolate (10-formyl-THF) to tetrahydrofolate (THF) and participates in the biosynthesis nucleotide precursor. It has been shown that FDH was down regulated during cell proliferation while over expression of FDH induced cell cycle arrest and apoptosis in cancer cells. Therefore the expression regulation of FDH is believed to tightly correlate to tumorgenesis and embryonic development. The aim of this study is to search for the possible mechanism of FDH expression regulation by cloning and characterizing the promoter region of zebrafish FDH (zFDH) gene. The 1703 bps fragment of zFDH promoter region was cloned upstream of luciferase coding sequence on pGL3-basic plasmid for transactivation activity analysis with Dual-luciferase assay in zebrafish liver epithelial cells (ZLE cell line). The 5’-UTR was cloned and the transcription starting site identified by PCR-cloning from the zebrafish full-length 5’-RACE cDNA library. Based on the results of serial deletion on promoter region and activity analysis, we found that the 164 bps (-124/+40) fragment is sufficient for substantial promoter activity. Deletion of the 40 bps 5’-UTR in exon 1 results in completely lost of promoter activity. The 164 bps fragment was DIG-labeled and incubated with nuclear extract in Electrophoresis gel Mobility Shift Assay (EMSA) and specific protein-nucleotide complexes were observed. Sequence analysis of the 164 bps fragment reveals potential binding site for Sp1 and c-Myb transcription factors. Site-directed mutagenesis to abolish these corresponding target sites results in decreased for Sp1 but no significant change for c-Myb transactivation activity. The coding sequence of zebrafish Sp1 had been cloned into the pET43.1a and pcDNA 3.1-myc/his expression vectors. We purified and characterized of the over-expressed Sp1 in E.coli and co-expressed the Sp1 in ZLE cells for promoter activity analysis.
論文目次 一、圖目錄 ………………………………3
二、表目錄 …………………………3
三、背景 …………………………4 ~ 11
四、研究動機 ……………… 12
五、研究材料及方法 ………………… 13 ~ 26
I. 材料 ………………………… 13
II. 斑馬魚肝臟上皮細胞與胚胎幹細胞的培養 …………13
III. zFDH 啟動子區域的序列分析 ………………… 13
IV. zFDH 啟動子區域的選殖 …………………… 14
V. zFDH 5’ UTR 全長序列的選殖 ……………… 14 ~ 15
VI. TA 選殖法 (TA cloning) ……………… 15 ~ 16
VII. zFDH 啟動子區域在reporter 載體的選殖與連續序列縮減 ……………… 16
VIII. pGL-3 載体選殖 …………………16
IX. zFDH啟動子-124/+40區域的單點突變 ……………17
X. ZLE細胞的轉染 …………………… 17 ~ 18
XI. 同時轉染與表現zFDH 啟動子-124/+40
和Sp1表現質體至ZLE細胞 ……………18
XII. 啟動子活性分析(Dual-luciferase assay) ……… 18 ~ 19
XIII. 斑馬魚肝臟與胚胎的核蛋白萃取 ………… 19
XIV. 凝膠遷移檢測
(Gel Mobility Shift Assay, GMSA) ………………19 ~ 21
XV. 斑馬魚Sp1短基因序列的選殖 …………… 21 ~ 22
XVI. 斑馬魚Sp1與c-Myb基因序列的選殖 …………22
XVII. 斑馬魚Sp1與c-Myb蛋白
(zSp1&zc-Myb)基因序列的表現………………… 23
XVIII. 斑馬魚Sp1蛋白的純化 …………………23 ~ 25
XIX. 西方墨點法(Western blot) ………………… 25 ~ 26
六、結果 ……………………… 27 ~ 30
七、討論 …………………… 31 ~ 32
八、參考文獻 …………………… 33 ~ 39
九、圖 ……………………… 40 ~ 55
十、表格 …………………… 56 ~ 60
十一、附件 …………… 61 ~ 96
十二、自述 ……………………… 97
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