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研究生:蓋鈺宸
研究生(外文):Yu-Chen Kai
論文名稱:探討雞毛囊中生殖激素的標的基因
論文名稱(外文):Characterization of Sex Hormone Target Genes in Chicken Feather Follicles
指導教授:鄭旭辰
指導教授(外文):Hsu-Chen Cheng
口試委員:鍾正明唐品琦
口試委員(外文):Cheng-Ming ChuongPin-Chi Tang
口試日期:2021-07-29
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:134
中文關鍵詞:性激素受體性激素受體調控序列羽毛性別兩型性報導基因活性分析
外文關鍵詞:sex hormone receptorsex hormone response elementsfeathersexual dimorphismreporter assay
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家雞 (gallus gallus domesticus)為台灣最常見之動物,同時也是分布最廣、家禽中數量最多的動物。因被人類長期馴化,使牠多樣的性狀累積,讓家雞成為遺傳學研究的重要模式動物。其雌雄個體具有性別兩型性,也就是在外觀、體型上有差異,其中也以羽毛最為顯眼,羽毛不只具有裝飾作用,同時也是生物表皮系統重要之模型。就外觀而言,雄性的羽毛成尖頭並且細長,也會因其品種而有不同的圖騰;而雌性的羽毛則呈圓頭也較短,顏色也較單一狀。在過去有許多研究關於性別兩型性之分子機制,前人研究中也以家雞作為材料,並利用次世代定序、生物資訊分析,以及Assay for Transposase-Accessible Chromatin using sequencing (ATACseq)之方法,找出可能影響性別兩型性的候選基因,但仍未有關鍵性之證據。

由於在前人研究中,利用生物資訊分析之方式,預測出雌激素調控序列 (estrogen response elements, EREs)以及雄激素調控序列(androgen response elements, AREs)所在位置,但在ATACseq分析中卻發現,不只有預測到ERE的位置有開放性差異-Wnt11,沒有預測到的位置也具有開放性之差異-OCA2,因此我們推測其中是否有非典型ERE參與其中。而有預測到的位置卻沒有開放性之差異-KITLG,推測為此片段有功能,但卻不是於In vivo狀態下之首選調控位點。在本試驗中,我們將其基因分組,設計序列引子,做報導基因 (Reporter assay)之活性分析;也將ATACseq做進一步分析成in silico footprinting,並與RNAseq的結果做比對,從中挑選出其基因具有性激素調控序列ERE- PTN、PTX3以及ARE- ABCA1、NREP,也將其序列設計引子,做報導基因測試 (Reporter assay)之活性分析。

結果顯示,在預測到ERE且ATACseq有差異的組別為有效片段;未預測到ERE但ATACseq有差異也為有效片段;有預測到ERE,但ATACseq卻未有差異的片段luciferase量也有提升,雖然在過高濃度的雌激素作用下,會導致細胞自主性調控。而利用in silico footprinting以及RNAseq所挑選出具有ERE的基因也為有效片段,但此片段對於雌激素相對較於敏感;以及ARE影響的基因,結果顯示由於閹割公雞血清內少量的雄性激素,而導致ARE並未受到雄性激素之調控,然而使用雄性激素受體抑制劑,可以有效降低其活性反應,藉此證明此片段為有效片段。由於前人研究顯示雌激素受體,對於羽毛的性別兩型性之重要性,因此於本試驗中也使用了雌激素受體抗體,了解雌激素受體在羽毛內的分布狀況以及狀態。


以上實驗我們得知了影響羽毛兩型性的關鍵基因,也從先前實驗得知雌激素受體的重要性。因此我們也設計了CRISPR/Cas9雌激素受體的質體,可以藉由基因編輯的方式,knockout雌激素受體,來知道若將雌激素受體敲除,會對於這些基因以及羽毛型態有何影響,為未來的研究提供下一步之開端。
Chicken (gallus gallus domesticus) is the most common animal in Taiwan, and it is also the most widely distributed and most abundant animal. Because its long-term domestication by humans, it has accumulated a variety of traits, making domestic chickens an important model animal for genetic research. The male and female individuals have sexual dimorphism, that differences in appearance and body shape. Especially feathers are the most conspicuous. Feathers are not only decorative, but also an important model of the integumentary system. In terms of appearance, male feathers are pointed and slender, and have different pattern depending on their strain; while female feathers are round and shorter, and their color is simpler. In the past, there have been many research on the molecular mechanism of sexual dimorphism. In previous studies, chickens were used as materials, and used the Next Generation Sequencing (NGS), bioinformatics analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATACseq), to identify candidate genes that may affect sexual dimorphism, but there is still no critical evidence.
In previous studies, the locations of estrogen response elements (EREs) and androgen response elements (AREs) were predicted using the bioinformatics analysis-Wnt11, but it was found in ATACseq analysis that unpredicted positions also have open chromatin pattern-OCA2, therefore we speculate there have non typical EREs or AREs. While predicted positions have no open chromatin pattern-KITLG, It is speculated that this fragment is functional, but it is not the preferred regulatory site in the In vivo. In this study, we grouped genes, designed sequence primers, and analyzed the activity of the reporter gene; we also further analyzed ATACseq into in silico footprinting, and compared them with the results of RNAseq to select the genes. The genes have sex hormone regulatory sequences ERE and ARE-PTN、PTX3、ABCA1、NREP, and their sequences are also selected out and inserted in plasmids for the activity analysis.
The results showed that the groups where EREs which were predicted and the ATACseq showed open chromatin, were effective fragments; the group of the EREs which were not predicted but the ATACseq showed open chromatin, were effective fragments too. And the EREs which were predicted but the ATACseq did’t show open chromatin, also showed the increase amount of luciferase, although cell-autonomous regulation in the high concentrations of estrogen seemed to happen.
The fragments of gene selected by in silico footprinting and RNAseq is an effective EREs fragments, but these fragments are relatively sensitive to estrogen. And genes affected by AREs may due to the less androgen remained in castrated rooster serum, lead to luciferase expression not be regulated by external androgen, but used the androgen receptor inhibitor could effectively reduce their active response, and proved their effectiveness.
Since previous studies have shown the importance of the estrogen receptor for the sexual dimorphism of feathers, so we also did the immunohistochemistry by estrogen receptor antibodies to understand the distribution and status of estrogen receptor in feathers.
In this study, we know the key genes to affected sexual dimorphism of feathers, and also confirmed the importance of estrogen receptor from previous experiments. Therefore, we also designed the plasmid of the CRISPR/Cas9 estrogen receptor for further analysis. We can use gene editing method to knockout the estrogen receptor, to know how it will affect these genes and feather patterns for future research.
摘要 i
Abstract iii
表次 viii
圖次 ix
前言 1
文獻探討 3
一.羽毛的構造 3
二.羽毛的性別兩型性 (Sexual dimorphism) 5
三.羽毛的發育(The development of feather) 6
四.性激素受體 8
4.1 雌性激素受體結構 8
4.2雌性激素受體作用機制 9
4.3雄性激素受體結構 9
4.4雄性激素受體作用機制 9
4.5性激素對於性別兩型性之影響 10
五.性激素受體標的基因 12
六. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) 15
目的與假說 17
一.實驗目的 17
二.實驗假說 17
三.實驗設計 18
材料與方法 20
一、實驗動物 20
二、實驗常用方法 20
2.1血液樣本採集 20
2.2毛囊樣本採集 20
2.3 DNA樣本萃取 20
2.4 RNA樣本萃取 21
2.5反轉錄PCR (Reverse transcription-PCR, RT-PCR) 21
2.6即時聚合酶連鎖反應 (Real-time polymerase chain reaction, qPCR ) 22
2.7羽毛組織固定、包埋 23
2.8免疫組織螢光染色 (Immunohistochemistry,IHC) 24
2.9雞纖維母細胞 (DF-1)培養&性激素加藥試驗 24
2.10 DNA膠純化 24
2.11 pGEMT TA clone與轉染 (Transformation) 25
2.12 pGL3質體構築 (Construct)與轉染 (Transformation) 26
2.13目標質體構築 26
2.14傳統抽取小量質體 (plasmid)DNA 27
2.15 Plasmid Miniprep Plus Purification kit 27
2.16抽取大量質體 28
2.17 DNA酵素截切 28
2.18電穿孔轉染 29
2.19 Lipofectamin3000轉染 30
2.20核蛋白萃取 (Nuclear extraction) 30
2.21 Luciferase reporter assay 31
三、ERE、ARE 目標基因分析 32
3.1 ATAC分析軟體 32
3.2序列比對軟體 32
3.3預測蛋白軟體 32
3.4 ATAC開放序列轉錄活性測試 (pGL3 luciferase reporter assay) 32
3.5 In silico footprinting分析軟體 33
四、引子設計 33
實驗結果 35
一、雌激素影響羽毛型態及候選標的基因 35
二、雌性激素受體標的基因調控序列活性分析 35
三、雄性激素受體標的基因調控序列活性分析 39
四、雌激素受體 (ESR1)在毛囊內表現及作用位置 41
4.1羽毛縱切第三天 42
4.2羽毛橫切第三天 42
4.3羽毛縱切第十天 43
討論 44
一、受ERE與ARE影響的標的基因 44
二、羽毛染色圖 45
三、CRISPR/Cas9 knockout ER 46
四、受雌激素影響的轉錄因子 46
結論 48
圖表 49
參考文獻 79
附錄 84
一、In silico footprinting ERE list 84
二、In silico footprinting ARE list 97
三、In silico footprinting ERE list v.s. RNAseq Castrate male & Castrate male E list 115
四、In silico footprinting ERE list v.s. RNAseq WT female & Spay female list 121
五、In silico footprinting ARE list v.s. RNAseq WT male & WT female list 126
六、In silico footprinting ARE list v.s. RNAseq WT male & Castrate male list 133
七、在雌激素處理後,ESR1改變了其結合位點與調控基因列表 133
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