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系統識別號 U0026-0812200915275159
論文名稱(中文) 尋找DAZL調控的下游基因並探討其單一核苷酸多型性對DAZL之功能影響
論文名稱(英文) To identify the target RNAs regulated by DAZL and characterize the effect of Single-Nucleotide Polymorphism on DAZL’s function
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 陳亭燕
研究生(英文) Ting-yen Chen
學號 s1696411
學位類別 碩士
語文別 中文
論文頁數 66頁
口試委員 指導教授-郭保麟
指導教授-曾大千
口試委員-孫孝芳
口試委員-張文粲
口試委員-張典顯
中文關鍵字 不孕症  單一核苷酸多型性  後轉錄調控作用 
英文關鍵字 NUDT21  DAZL  solexa  post-transcriptional regulation  infertility  SNP 
學科別分類
中文摘要 根據世界衛生組織(World Health Organization)統計,約15%的夫婦受到不孕症的影響,在男性不孕症病人常見其Y染色體長臂上有片段的缺失,此片段為AZF區域包含DAZ基因,DAZ的同源基因DAZL位於三號染色體,會專一性表現於生殖細胞,是一個含有RNA-recognition motif與DAZ repeat的RNA binding protein,在DAZL基因剔除的動物模式實驗發現會造成生殖細胞的缺失,文獻也指出DAZL會結合不同基因的mRNA進行後轉錄作用的調控,藉由促進不同基因之mRNA的轉譯作用在生殖細胞精子形成過程中行使不同的功能,顯示DAZL在生殖細胞的發育與分化扮演重要的角色。實驗室之前在患有無精子症與少精症的台灣男性不孕症病人篩選到數個核苷酸突變與單一核苷酸多型性的變異,特別是在核苷酸386 (T54A) 位置由adenine變成guanine,此單一核苷酸多型性在不孕症病人較為盛行。為了闡明單一核苷酸多型性對DAZL功能的影響,首先我們先利用RNA免疫沉澱分析並結合deep-sequencing技術篩選DAZL可能作用的mRNA,得到NUDT21、NUP50、KLHL12、KDELR1、VAPA、ABHD2、SUPT5H、MLL3、ATP6AP2等可能是DAZL調控的transcripts,其中NUDT21在文獻中指出會高量表現在老鼠生殖細胞且參與在精子形成過程中的alternative polyadenylation的機制,因此選定NUDT21作為探討DAZL調控機制的標的。利用核酸醣體免疫沉澱分析,結果顯示DAZL透過後轉錄機制調控NUDT21的表現,藉由抑制NUDT21的轉譯作用在生殖細胞扮演重要的角色,但當T54A單一核苷酸多型性DAZL大量表現時會降低與NUDT21的結合作用,因而無法抑制NUDT21的轉譯作用,顯示生殖細胞的DAZL可能會與不同蛋白質結合,來調控不同的基因參與精子形成過程的作用,而單一核苷酸多型性可能會影響與DAZL作用的結合蛋白,進而影響DAZL與下游RNA的結合能力,在後轉錄作用層面進行不同的調控。
英文摘要 Infertility affects 15% of all couples. Deleted in Azoospermia (DAZ) gene was isolated from the AZFc region of the human Y chromosome that is frequently deleted in infertile men. DAZ-like (DAZL) is an autosomal homolog located on chromosome 3p24. In human beings, DAZL expresses in germ cells with different developmental stages, from spermatogonia, meiotic spermatocytes to mature spermatozoa. Although DAZL is essential for spermatogenesis, the exact function remains undefined. It has been proposed that DAZL binds with mRNA expressed in germ cells and control spermatogenesis at the level of translation. However, specific target mRNAs regulated by DAZL in vivo are not clear. Using RNA immunoprecipitation combined with high-throughput sequence analysis, a number of target transcripts, including NUDT21, NUP50, KLHL12, KDELR1, VAPA, ABHD2, SUPT5H, MLL3 and ATP6AP2, were identified. Among these transcripts the regulation of NUDT21, one of the CFIm subunit, by DAZL was studies. NUDT21 are highly expressed in mouse male germ cells and involve in alternative polyadenylation directed by noncanonical poly(A) signals during spermatogenesis. Previously, we have identified several mutations and single nucleotide polymorphisms (SNP) of DAZL, which associated with spermatogenic failure. To characterize the functional role of DAZL polymorphisms or mutations, RNA-IP and S6-IP were used to examine the binding activity and translational efficiency of NUDT21, respectively. The binding activity was increased while the translational efficiency was decreased in the existence of normal DAZL. In contrast, the polymorphism of DAZL, A to G transition at nucleotide 386, was associated with decreased binding activity and increased translational efficiency for NUDT21. These data support NUDT21 as a candidate target of DAZL-mediated translation and suggest that DAZL may indirectly participate in alternative polyadenylation during spermatogenesis.
論文目次 中文摘要 i
英文摘要 ii
謝誌 iii
目錄 iv
表目錄 vi
圖目錄 vii
附錄 viii

第一章 序論 1
第一節 男性不孕症 1
第二節 精子形成過程 1
第三節 DAZ基因家族在男性不孕症的角色 2
第四節 DAZL在精子形成過程的角色 3
第五節 DAZL的功能 4
1.5.1 DAZL參與後轉錄基因的調控 4
1.5.2篩選DAZL調控的target messenger RNA substrate之方法 5
1.5.3 DAZL調控的target messenger RNA substrate 5
1.5.4 DAZL調控的target messenger RNA substrate序列 7
1.5.5 DAZL參與後轉錄調控作用的機制 7
1.5.6 DAZL的結合蛋白 8
第六節 系統生物學的運用 8
1.6.1系統生物學 8
1.6.2轉錄體 9
1.6.3 RNA-Seq技術 10
第七節 研究動機 11
第二章 材料方法 12
實驗材料 12
實驗方法 17
第一節 細胞培養 17
2.1.1人類子宮頸癌 (HeLa) 細胞之培養 17
2.1.2短暫性轉移感染 17
第二節 分析RNA的表現 17
2.2.1 RNA抽取 17
2.2.2反轉錄反應 18
2.2.3聚合酶連鎖反應 19
2.2.4定量PCR 19
第三節 分析蛋白質的表現 20
2.3.1製備cell lysate 20
2.3.2蛋白質濃度測定 21
2.3.3西方點墨法 21
第四節 質體的建構 21
2.4.1小量質體置備 21
2.4.2大量質體置備 22
2.4.3 DNA膠體純化 22
2.4.4 DNA/RNA純化 23
2.4.5限制酶處理 23
2.4.6 DNA接合 24
2.4.7轉形作用 24
第五節 RNA免疫沉澱分析 24
第六節 Deep sequencing樣品製備 25
2.6.1 GeneRacer樣品製備 25
2.6.2 solexa樣品置備 26
第七節 核酸醣體免疫沉澱分析 27
第八節 電腦軟體及統計分析 27
第三章 實驗結果 28
第一節 選定RNA-IP和RNA-Seq探討DAZL調控的下游基因 28
第二節 確認被DAZL調控的下游基因 28
第三節 選定NUDT21作為被DAZL調控的下游基因 29
第四節 DAZL會結合在NUDT21的mRNA 30
第五節 DAZL在後轉錄過程調控NUDT21扮演的角色 30
第六節 探討單一核苷酸多型性對DAZL功能之影響 31
第四章 討論 32
第一節 deep sequencing結果分析 32
第二節 NUDT21在精子形成過程扮演的角色 33
第三節 DAZL在精子形成過程參與的後轉錄調控機制 35
第四節 其他可能被DAZL調控的基因 36
第五節 總結 37
參考文獻 38
表 46
圖 52
附錄 65
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