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研究生:藍丹穗
研究生(外文):Dan-Suei Lan
論文名稱:nr5a1a和nr0b1在黑鯛性轉變過程中對性類固醇合成過程相關基因的調控關係
論文名稱(外文):The interaction of nr5a1a and nr0b1 in steroidogenic genes during sex change in protandrous fish, Acanthopagrus schlegeli
指導教授:張清風張清風引用關係
指導教授(外文):Ching-Fong Chang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:73
中文關鍵詞:黑鯛性類固醇合成相關基因
外文關鍵詞:black porgysteroidogenic genesnr5a1a(sf-1)nr0b1(dax-1)
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黑鯛為先雄後雌之雌雄同體海水魚類,在一到兩年齡為功能性雄性,在第三年可能性轉變為雌性。本研究之目的在探討黑鯛性別發育及性轉變時期,其性別相關調控基因之間的關係。主要是探討nr5a1a (steroidogenic factor 1, sf-1) 和nr0b1 (dosage sensitive sex-reversal, adrenal hypoplasia congenita critical region on the X-chromosome, gene 1, dax-1) 在黑鯛性別發育及性轉變過程中對性類固醇合成過程相關基因的調控關係。利用基因即時定量系統分析測定nr5a1a、nr0b11及性類固醇合成相關基因在黑鯛之生殖腺表現量,比較並分析其對於生殖腺發育及彼此之間相關調控關係。結果發現在精巢組織,nr5a1a基因在1+~2+黑鯛表現量高,nr0b1基因表現量在1+~2年齡黑鯛發生精子生成時期高但至2+黑鯛低,其蛋白質表現分布在精原細胞,而部分性類固醇合成相關基因 (star、cyp11a1及cyp11b2) 在1+~2+黑鯛精巢表現量高。推測nr5a1a及部分性類固醇合成相關基因與精巢發育活化可能相關,而nr0b1與精子生成作用相關。在卵巢組織,nr0b1基因表現量高,蛋白質表現分布在卵原細胞,nr5a1a及部分性類固醇合成相關基因表現量低。推測nr0b1可能與未活化卵巢組織發育相關,且與nr5a1a及部分性類固醇合成相關基因為負相關。cyp19a1a基因在黑鯛卵巢組織中表現量高,與卵巢組織發育可能相關。
Black porgy, Acanthopagrus schlegeli, a marine protandrous hermaphrodite fish, is functionally male for the first and second year of life, but begins to sexually change to female after the third year. The objectives of the study were to investigate the expression of the sex-related genes. The major study was to investigate the interaction of nr5a1a (steroidogenic factor 1, sf-1) and nr0b1 (dosage sensitive sex-reversal, adrenal hypoplasia congenita critical region on the X-chromosome, gene 1, dax-1) in steroidogenic genes during sex development and sex change in black porgy. Quantification of nr5a1a, nr0b1 and steroidogenic genes transcripts were conducted by real-time PCR. nr5a1a transcripts were high in testicular tissue in 1+ to 2+-yr-old black porgy. nr0b1 transcripts were high during spermatogenesis in 1+ to 2-yr-old black porgy but were low in 2+-yr-old black porgy, and Nr0b1 was expressed in spermatogonia. Some of the steroidogenic genes( star, cyp11a1 and cyp11b2) transcripts were low in testicular tissue in 1+ to 2+-yr-old black porgy. nr5a1a and some of steroidogenic genes may be related to testicular tissue development and nr0b1 may be related to spermatogenesis. nr0b1 transcripts were high in ovarian tissue and Nr0b1 was expressed in oogonia. nr5a1a and some of steroidogenic genes transcripts were low in ovarian tissue. nr0b1 may be related to development of inactive ovarian tissue and its relation with nr5a1a and some of steroidogenic genes were negative. cyp19a1a transcripts were high in the ovarian tissue, it may be related with development of ovarian tissue.
謝辭……………………………………………………………………i
中文摘要………………………………………………………………ii
英文摘要……………………………………………………………iii
目錄……………………………………………………………… iv
圖表目錄…………………………………………………………… vii


第一章 諸論………………………………………………………………1
1、性別分化與發育………………………………………………………1
(1) 哺乳動物的性別決定…………………………………………1
(2) 硬骨魚類的性別發育………………………………………2
2、影響魚類性別的相關因子……………………………………………3
(1) 內分泌系統 ……………………………………………………3
(2) nr5a1a…………………………………………………………5
(3) nr0b1……………………………………………………………7
3、研究緣起………………………………………………………………8
4、研究目的………………………………………………………………9

第二章 基因即時定量系統之建立……………………………………10
1、前言……………………………………………………………………10
2、實驗方法………………………………………………………………10
(1) 即時定量Real-time PCR系統建立…………………………10
3、實驗藥品………………………………………………………………14
(1) 菌種、質體…………………………………………………14
(2) 一般化學藥品…………………………………………………15
(3) 酵素及生化分析試劑………………………………………15
(4) 實驗藥品及溶液配方…………………………………………16
(5) 統計分析………………………………………………………17
4、結果…………………………………………………………………18
(1) 即時定量Real-time PCR系統之建立………………………18
5、討論…………………………………………………………………19
(1) 黑鯛基因標準品製備.………………………………………19
(2) 反應值回歸…………………………………………………19
圖……………………………………………………………………20

第三章 1+~3年齡黑鯛在雄性階段與性轉變時期nr5a1a、nr0b1及性類固醇合成相關基因之季節性變化………………………………………24
1、前言…………………………………………………………………24
2、實驗設計與方法……………………………………………………25
(1) 實驗設計……………………………………………………25
(2) 實驗方法………………………………………………26
3、結果………………………………………………………………28
(1) 1+~2年齡雄性黑鯛成為功能性雄性過程nr5a1a、
nr0b1基因在生殖腺組織的表現…………………………………………28
(2) 1+~2年齡黑鯛之Nr5a1a及Nr0b1蛋白質在精巢
組織與卵巢組織之分布……………………………………29
(3) 1+~2年齡雄性黑鯛成為功能性雄性各類固醇合成
相關基因在生殖腺組織的表現………………………………29
(4) 比較3年齡黑鯛維持雄性功能或性轉變之2+黑鯛
非繁殖季生殖腺組織nr5a1a、nr0b1以及各類固醇合成
相關的基因表現……………………………………………………31
4、討論…………………………………………………………………33
圖………………………………………………………………………36

第四章 結論……………………………………………………………65

參考文獻………………………………………………………………66

圖表目錄
圖1、黑鯛Star基因即時定量系統之建立……………………………20
圖2、黑鯛Cyp11a1基因即時定量系統之建立………………………21
圖3、黑鯛Cyp17a1基因即時定量系統之建立………………………22
圖4、黑鯛Cyp19a1a基因即時定量系統之建立………………………23
圖5、1+~2年齡雄性黑鯛成為功能性雄性過程
生殖腺組織nr5a1a之基因表現…………………………………………36
圖6、1+~2年齡雄性黑鯛成為功能性雄性過程
生殖腺組織nr0b1之基因表現…………………………………………37
圖7、1+黑鯛生殖腺組織Nr0b1免疫染色……………………………38
圖8、1+~2年齡雄性黑鯛成為功能性雄性過程
生殖腺組織star之基因表現………………………………………………39
圖9、1+~2年齡雄性黑鯛成為功能性雄性過程………………40
圖10、1+~2年齡雄性黑鯛成為功能性雄性過程
生殖腺組織cyp17a1之基因表現………………………………41
圖11、1+~2年齡雄性黑鯛成為功能性雄性過程
生殖腺組織cyp19a1a之基因表現……………………………42
圖12、1+~2年齡雄性黑鯛成為功能性雄性過程
生殖腺組織cyp11b2之基因表現………………………………43
圖13、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季生殖腺組織的nr5a1a表現…………………………44
圖14、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季生殖腺組織的nr0b1表現…………………………45
圖15、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季生殖腺組織的star表現……………………………46
圖16、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季生殖腺組織的cyp11a1表現………………………47
圖17、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季生殖腺組織的cyp17a1表現………………………48
圖18、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季生殖腺組織的cyp19a1a表現………………………49
圖19、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季生殖腺組織的cyp11b2表現………………………50
圖20、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季精巢組織的nr5a1a表現…………………………51
圖21、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季卵巢組織的nr5a1a表現…………………………52
圖22、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季精巢組織的nr0b1表現…………………………53
圖23、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季卵巢組織的nr0b1表現……………………54
圖24、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季精巢組織的star表現……………………………55
圖25、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季卵巢組織的star表現………………………56
圖26、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季精巢組織的cyp11a1表現…………………57
圖27、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季卵巢組織的cyp11a1表現…………………………58
圖28、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季精巢組織的cyp17a1表現………………59
圖29、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季卵巢組織的cyp17a1表現……………………………60
圖30、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季精巢組織的cyp19a1a表現…………………………………61
圖31、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季卵巢組織的cyp19a1a表現………………………………62
圖32、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季精巢組織的cyp11b2表現……………………………………63
圖33、比較3年齡黑鯛維持雄性功能或性轉變之
2+黑鯛非繁殖季卵巢組織的cyp11b2表現……………………………………64
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