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博碩士論文 etd-0917109-121938 詳細資訊
Title page for etd-0917109-121938
論文名稱
Title
探討HINT1與HINTW對WDR36近端啟動子區域之調控
The HINT1 and HINTW responsive element(s) in WDR36 proximal promoter region
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
61
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2009-07-24
繳交日期
Date of Submission
2009-09-17
關鍵字
Keywords
性別決定、cDNA基因庫、雞
WDR36, sex determination, HINTW, HINT1, cDNA library, Chicken (Gallus gallus)
統計
Statistics
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中文摘要
關於鳥類性別發育與分化時的決定性因素,目前存在有兩種假說:一是由於性染色體的不平衡,即依照基因劑量 (Z-chromosome dosage)來主導;另一則是由雌性禽類 (ZW) 特有的性染色體『W chromosome』來主導。在先前的研究裡,本研究室建構了在生殖腺外觀發育前,差異性表現於雄性及雌性胚胎的雌性減除雄性cDNA庫 (Female-subtract-male cDNA library),並且辨識了 279 個可能差異性高表現於雌性個體的表現序列 (expression sequence taq;EST)。進一步採用定量反轉錄聚合酵素鏈鎖反應 (quantitative RT-PCR) 技術,分析其中 16 個表現序列及三個經報導參與雞胚胎性別發育的標記基因HINT1、FET-1及WDR36在胚胎早期(第三、五、七、九天)不同性別間的表現情形。結果顯示AGR2、CPT2、DUSP19、HINTW、LOC771368及EY53070791在分析早期(第三與第五天),於雌性胚胎表現顯著高於雄性;FET-1於雌性的表現量則隨著分析時間的推進逐漸顯著上升。另外,HINT1與WDR36皆在雄性胚胎表現高於雌性胚胎,然HINT1在胚胎發育早期有較高的表現量,而WDR36則在所分析時間的晚期(第七與九天)有顯著較高表現。利用雞B細胞細胞株DT40過量表現融合GFP螢光蛋白的 HINT1,使得WDR36 mRNA表現量上升;相反的,過量表現GFP-HINTW融核蛋白,則導致WDR36 mRNA表現量下降。進一步用RNA干擾(RNA interference)技術降低HINTW表現,結果顯示,WDR36轉錄子表現上升;最後,我們分析過量表現HINT1-GFP融合蛋白對進WDR36近端啟動子的調控情況,證實了HINT1-GFP融合蛋白可促進WDR36近端啟動子的表現。因此,我們推測HINT1蛋白質藉由影響位在近端的啟動子序列來調控WDR36轉錄子表現。未來應進行深一步的實驗探討HINT1及HINTW是藉由直接或間接的影響,來對WDR36啟動子區域進行調控。
Abstract
Two hypotheses currently exist regarding to the determining factors for sexual development and differentiation in birds. One is based on the unbalanced sex chromosome, meaning that avian sex determination is dominated by “Z-chromosome dosage”. The other brings up (reconsider this) the key factor of “W chromosome” which is a particular sex chromosome in female birds (ZW). In the previous studies, we constructed a female-subtract-male cDNA library before morphological gonad differentiation. After sequencing and annotation, a total of 279 expression sequence taqs (ESTs) were identified, with potentially higher expression levels in females. By utilizing quantitative RT-PCR, 16 potential ESTs and three marker transcripts (HINT1, FET1 and WDR36), which identified to be involved in sexual development at 3, 5, 7, 9 days post-coitum (dpc) was analyzed in chicken embryos. Results indicated that AGR2, CPT2, DUSP19, HINTW, LOC771368 and EY53070791 had higher expression levels in female than in male embryos at 3 and 5 dpc; FET1 expression level in female embryos gradually increased from 3 to 9 dpc. Moreover, both HINT1 and WDR36 were higher expressed in male than in female embryos across 3 to 9 dpc. However, HINT1 exhibited higher expression levels starting at early stage, whereas WDR36 at later stage. Next, we constructed HINT1-GFP fusion protein and overexpressed this protein in chicken B-cell line (DT40), resulting in upregulation of WDR36 expression. On the contrary, overexpressed HINTW-GFP fusion protein in DT40 cells had decreased WDR36 expression level. Moreover, we designed a small hairpin RNA by utilizing RNA interference technique to knockdown expression of HINTW, which resulted in WDR36 upregulation. Finally, we then estimated the regulation of WDR36 promoter activity through analyzing HINT1-GFP overexpression. Results had shown that HINT1-GFP can improve WDR36 promoter activity. Therefore, we suppose that HINT1 can regulate WDR36 transcription via WDR36 proximal promoter region. Ongoing HINT1 responsive element(s) must be identified to characterize whether HINT1 or HINTW regulates WDR36.
目次 Table of Contents
Contents
Abstract in Chinese ---------------------------------------------------------- I
Abstract in English ---------------------------------------------------------- II
Abbreviation ----------------------------------------------------------------- III
Introduction ------------------------------------------------------------------ 1
Materials and Methods ----------------------------------------------------- 11
Results ------------------------------------------------------------------------ 16
Discussions ------------------------------------------------------------------- 21
Figures and tables ----------------------------------------------------------- 24
Supplementary data --------------------------------------------------------- 40
Appendix --------------------------------------------------------------------- 45
References ------------------------------------------------------------------- 50
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