生產鴨用或肉用雄性番鴨，係為農牧業的重要經濟目標之一。儘管目前對性別決定機制迄今尚未闡明，操縱家禽性別決定基因，將可供發展選性繁殖系統之用。果蠅DSX、線蟲MAB-3基因與人類、鼠科動物、雞隻、魚類、海龜與短吻鱷性別決定基因DMRT1，在演化上顯示顯示具高度保留性。因此，雞隻性染色體Z連鎖之DMRT1基因，成為家禽調控性別分化之候選調節基因。本論文目的要選殖並瞭解番鴨DMRT1基因，以進一步應用於選性繁殖上。目前以定序出番鴨Muscovy DMRT1 cDNA部分序列，並顯示番鴨DMRT1 cDNA序列與雞隻及海龜分別有95%及83%相同度，而番鴨DMRT1胺基酸序列則與雞隻及海龜分別有61%及54%相同度。以Phylip程式分析各物種間之DMRT1異物種同源基因並以MEGA 2程式繪製親緣樹。以上結果顯示家禽DMRT1基因在cDNA序列與胺基酸序列層次上之親緣關係，均與爬蟲類較相近，而與哺乳類有較遠的親緣關係。

To produce male Muscovy only for fatty liver and meat-type production is an important economic goal in animal husbandry, although the sex-determining mechanism in poultry remains to be elucidated. Manipulation of sex-determining gene(s) in poultry provides enormous opportunities on the development of sex pre-selection reproductive systems. DSX and MAB-3 genes in Drosophila and C. elegans are conserved across the human, mice, chickens, fish, turtles, and reptiles revealing an ancient sex-determining locus DMRT1. Thus the Z-linked, DMRT1 in chicken is an excellent candidate regulatory gene controlling similar aspects of sexual development in poultry. This dissertation is aimed to clone and characterize Muscovy DMRT1 gene for further application in sex pre-selection. Partial cDNA sequences of Muscovy DMRT1 was determined and revealed 95% identity and 83% with chicken and red-eared slider turtle DMRT1 cDNA sequences. DMRT1 orthologs among various species were analyzed by Phylip program and phylogenetic tree was constructed by MEGA2 programs. Results indicated that Muscovy, chicken and red-eared slider turtle DMRT1 revealing 95%, and 83% identity at cDNA and 61%, 54% identity at amino acid level.

中文摘要.....................................................................................................Ⅰ
英文摘要.....................................................................................................Ⅱ
英文縮寫表.................................................................................................Ⅲ
論文正文
第一章序言.............................................................................................1
第二章文獻檢討
2.1 家禽的選性繁殖................................................................3
2.2 鳥類之基因組....................................................................3
2.3 性染色體............................................................................4
2.3.1 哺乳類性染色體......................................................5
2.3.2 鳥類的性染色體......................................................5
2.3.3 鳥類性染色體與性別決定基因的演化..................6
2.4 性別決定的假說................................................................6
2.4.1 遺傳因子平衡模式..................................................7
2.4.2 顯性基因模式.........................................................7
2.5 家禽之早期性別鑑定.........................................................8
2.6 參與家禽性腺發育之基因.................................................9
2.7 W性染色體連鎖基因，Wpkci..........................................11
2.8 DMRT1
2.8.1 DMRT1異物種同源基因.......................................12
2.8.1.1線蟲MAB-3與果蠅DSX ....................................13
2.8.1.2 人類DMRT1.......................................................13
2.8.1.3 雞隻DMRT1.......................................................14
2.8.2 人類9p 缺失與DMRT1......................................14
2.8.3 比較基因定位.......................................................15
2.8.4 人類DMRT1 基因之基因結構............................15
2.9 溫度調控爬蟲類之性別決定..........................................16
2.10 番鴨................................................................................17
第三章材料與方法
3.1 矽晶選殖番鴨性別決定候選基因DMRT1.....................18
3.1.1 由GenBank 與表現序列標幟資料庫搜尋番鴨
DMRT1的異物種同源基因................................18
3.1.2 引子設計...............................................................18
3.2 分子選殖番鴨DMRT1 基因
3.2.1 番鴨.......................................................................19
3.2.2 萃取睪丸組織的total RNA..................................20
3.2.3 反轉錄-聚合? 鏈鎖反應......................................20
3.2.4 熱啟動聚合? 鏈鎖反應........................................21
3.2.5 TA cloning 與大腸桿菌轉殖作用.........................22
3.2.6 自瓊脂凝膠純化DNA 片段................................23
3.2.7 5' Rapid Amplification of cDNA Ends..................24
3.2.8 3’ Rapid Amplification of cDNA Ends..................25
3.2.9 定序.......................................................................26
3.2.10 序列分析與二級結構預測.................................27
第四章結果
4.1 矽晶選殖番鴨性別決定候選基因DMRT1.....................30
4.2 分子選殖DMRT1.............................................................31
4.3 DMRT1異物種同源基因之親緣樹..................................32
4.4 DMRT1 之二級結構預測.................................................33
第五章討論...........................................................................................34
第六章結論...........................................................................................37
參考文獻.....................................................................................................38
表
表一、不同物種性別發育過程中，基因型與外表型間的相對關係.....48
表二、番鴨的生活習性...........................................................................49
表三、PCR 反應所使用之引子對與PCR 反應之進行條件..................50
表四、雞隻DMRT1DNA 序列，經由blastn 檢索nr database，未發現
其他鳥類的DMRT1 異物種同源基因.......................................51
表五、以雞隻DMRT1 基因(Acc#AF123456)當作探針，所得異物種
同源序列之長度、一致性區域及百分比...................................52
表六、由資料庫所檢索DMRT1 異物種同源序列之長度、轉譯區，與
雞隻DMRT1 蛋白質序列間的相同度與相似性.......................53
表七、多重序列組合所分析之DMRT1 異物種同源基因列表...........54
表八、不同物種間DMRT1 胺基酸序列之相似性與相同度...............55
圖
圖一、雞隻染色體Z、W 上的基因，與人類第五、八、九、十八體
染色體的異物種同源基因.........................................................56
圖二、參與脊椎動物生殖腺發育特異階段之基因..............................57
圖三、Wpkci 與雞隻PKCI(chPKCI)之胺基酸序列比對.....................58
圖四、DM domain 之圖示(a)平面(b)立體帶狀結構..........................59
圖五、DMRT1 之基因結構..................................................................60
圖六、DMRT1異物種同源基因的生物資訊研究................................61
圖七、選殖番鴨DMRT1 基因之實驗流程圖..................................62
圖八、人類與雞DMRT1 cDNA 序列的保留性區域.........................63
圖九、番鴨DMRT1之PCR產物與雞隻DMRT1間之相對位置.........64
圖十、PCR 產物A、D、H、I、N之電泳分析圖.............................65
圖十一、番鴨DMRT1 基因列之核? 酸及蛋白質序列.....................66
圖十二、以Phylip 程式比較不同物種之DMRT1 胺基酸序列.........67
圖十三、不同物種間DMRT1基因序列之親緣樹
(a)ME (b)MP........................................................................71
(c)NJ (d)UPGMA................................................................72
圖十四、DM domain 之演化高度保留性.............................................73
附錄.............................................................................................................74

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