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研究生:許復傑
研究生(外文):Fu Jay Hsu
論文名稱:研究PLK基因mRNA穩定性調控機制
論文名稱(外文):Regulatory mechanism of PLK gene family mRNA stability
指導教授:王子豪王子豪引用關係陳淑貞陳淑貞引用關係
指導教授(外文):T. H. WangS. J. Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:58
中文關鍵詞:mRNA穩定性調控機制
外文關鍵詞:PLK3
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messenger RNA (mRNA)的穩定性在基因的表現當中扮演著非常重要的角色,mRNA的穩定性控制著基因表現量的多寡,我們想要藉由整體基因上的層面以及microarray的實驗數據來研究mRNA的穩定性。而經由microarray的實驗數據顯示,PLK基因的家族mRNA相對於其他基因的mRNA之下較為不穩定,而mRNA的穩定性受到很多機制調控,大約10%的哺乳動物的mRNA富含有腺嘌呤以及尿嘌呤,稱為AU-rich element (ARE),而且AU-rich element (ARE)可以影響mRNA的穩定性,我們分析了PLK基因序列的3’ untranslated region(3’UTR),以及確認是否有ARE存在於PLK基因的3’UTR上,我們也找到了一些PLK基因mRNA上可能的miRNA結合位置。而PLK基因群中的PLK3基因是含有ARE以及miRNA可能的結合位置,PLK3在細胞生理中主要是參與細胞週期以及DNA損傷時有相關。在此研究中,我們克隆出PLK3 3’UTR且證明了PLK3 3’UTR可以影響mRNA的穩定性。
Stability of messenger RNA(mRNA) is very important to control the steady state level of gene expression. Modulation of mRNA stability regulates gene expression level. We want to invest RNA stability by genome-wide analysis by microarray.Microarray data shows Plk families have relative unstable mRNA stability.Many mechanisms change mRNA stability. However, almost 10% of mammalian mRNAs contain adenine and uridine residues known as AU-rich element (ARE) , it can affect mRNA stability. We analyze Plk families 3’UTR and identify ARE motif in Plk3. We also find miRNA targets in Plk3.Plk3, one of Polo-like kinase family members, is involved in the regulation of cell cycle progression and DNA damage response. In this study,we clone Plk3 3’UTR which contains ARE and miRNA binding sites.We also clone Plk 3’UTR which contains ARE only.We determined Plk3 3’UTR controls its RNA stability.ARE determined Plk3 RNA stability.
指導教授推薦書………………………………………….………..…….i
口試委員審定書…………………………………………………………ii
授權書…………………………………………………………………….……… iii
誌謝……………………………………………………………………iv
英文摘要………………………………………………………………v
中文摘要………………………………………………………………vi
目錄……………………………………………………………………vii
第一章前言……………………………………………………………1
1.1 RNA stability……………………………………………………….1
1.2 Plk FAMILY………………………………………………………..5
第二章實驗動機………………………………………………………7
第三章材料方法………………………………………………………8
3.1細胞株培養………………………………………………………8
3.2 RNA抽取 …………………………………………………………8
3.3 DNase I處理………………………………………………………9
3.4反轉錄聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction, RT-PCR) ………………………………………………………9
3.5同步即時定量聚合酶連鎖反應 ( Quantitative real-time polymerase chain reaction, Q-PCR)……………………………………………….10
3.6生物晶片分析…………………………………………….………10
3.7報導蛋白表現分析(Reporter assay)…………………………11
3.8生物資訊分析……………………………………………………12
第四章 實驗結果……………………………….…………….………13
4.1測試ActD抑制DNA轉錄mRNA的適當濃度並以Q- PCR測試mRNA半衰期…………………………………………………………13
4.2 A549細胞的基因mRNA半衰期分析……………………………13
4.3 PLK基因Q-PCR Primer sequence設計…………………………14
4.4利用Q-PCR測試準確的PLK基因的半衰期……………………14
4.5找尋PLK基因family資料並挑選出有興趣的PLK3基因……15
4.6在HEK293細胞株中利用Reporter assay測試Luciferase activity
…………………………………………………………………………15
4.7 PLK3 3’UTR的ARE影響PLK3 mRNA穩定性………………17
第五章 討論……………………………………………………………19
附圖……………………………………………………………………22
附表……………………………………………………………………36
參考文獻………………………………………………………………41
附錄……………………………………………………………………46
參考文獻………………………………………………………………41
附錄……………………………………………………………………46
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