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研究生:謝和叡
研究生(外文):Huo-Jui Hsieh
論文名稱:腎型真葉珊瑚生殖腺發育成熟相關基因之研究
論文名稱(外文):Studies on the gonad development and maturation-related genes in the scleractinia coral, Euphyllia ancrora
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:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:81
中文關鍵詞:腎形珊瑚熱休克蛋白70促性腺激素釋放激素性類固醇
外文關鍵詞:coralHSP70GnRHsex steroids
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本論文的目的在探討腎形珊瑚(Euphyllia ancora)熱休克蛋白70與促性腺激素釋放素(GnRH)對生殖腺發育成熟可能的調控與機制。本研究利用 cDNA library 選殖到兩種腎形真葉珊瑚似熱休克蛋白70(heat shock protein 70,HSP70)基因片段,暫命名為 hsp70a 及 hsp70b 。熱休克蛋白70 在脊椎動物中與熱緊迫反應與配子生成有關,在無脊椎動物中與環境緊迫和配子發育有關,但在腎形珊瑚中是否有相關的機制仍不清楚,本研究進一步探討hsp70a與hsp70b基因在珊瑚生殖腺發育成熟時的表現與相關性。本論文發現雄珊瑚與雌珊瑚的 hsp70a 基因在繁殖季與非繁殖季時基因表現皆無明顯差異,但在雌雄性別上有明顯差異,雌珊瑚的 hsp70a 基因表現較雄珊瑚高。雄珊瑚的 hsp70b 基因在非繁殖季時表現量高於繁殖季,雌珊瑚的 hsp70b 基因在繁殖季時表現量會上升,推測與卵細胞的發育有關。另外本論文利用生殖腺細胞培養方式追蹤 「3H」Pregnenolone 在生殖腺成熟過程之代謝產物,探討GnRH對珊瑚生殖腺發育成熟的調控機制。研究發現在珊瑚生殖腺組織可偵測到 11KT、20βS、S、E2、AD、DOC&T、17OHP、DHP、P5 性類固醇賀爾蒙代謝產物的存在,並經LHRH-A處理後雄珊瑚精巢的 11-ketotestosterone 濃度會增加,雌珊瑚卵巢的 Estradiol 、 Estrone和Androstenedione 濃度會增加,顯示 GnRH 對珊瑚生殖腺有直接的調控作用,推測珊瑚應具有相當完整的性類固醇生合成機制。
The objectives of this study were to investigate the heat shock protein 70(Hsp70)and Gonadotropin-releasing hormone (GnRH)in scleractinian coral, Euphyllia ancora. The possible roles of Hsp70 and GnRH in the gonad development and maturation of coral were also studied. By applying “cDNA library construct” were cloned two heat shock protein 70-like genes fragment(hsp70-like) in Euphyllia ancora, and temporarily to named hsp70a and hsp70b. Heat shock protein 70(Hsp70)was stress and gametogenesis-related gene in vetebrate and invertebrate, but the possible roles of heat shock protein 70 in coral were not clear. The objectives of were to investigate the expression and relationship of Hsp70 in the gonad development and maturation of coral. In the study, hsp70a in female and male had no significantly change from non-spawning season to spawning season, but female presented higher expression of hsp70a than male. In male corals the expression of hsp70b decreased from non-spawning season to spawning season, but in female corals the expression of hsp70b increased from non-spawning season to spawning season. It’s relationship with oogenesis. Another the study by the cultured of gonad cell to track to metabolic products of 「3H」Pregnenolone during gonad maturation, and the objectives of were to investigate the possbible roles of GnRH in the gonad development and maturation of coral. In the study, we found the sex steroid hormone metabolic products of 11KT、20βS、S、E2、AD、DOC&T、17OHP、DHP、P5 , and in male gonad tissue the concentration of 11-ketotestosterone increased by LHRH-A treated. In female ovary the concentration of Estradiol 、 Estrone和Androstenedione increased by LHRH-A treated. GnRH had the directly regulative function to the coral gonad, and extrapolated the coral should have the completely mechanism of sex steroidogenesis.
目錄
頁次
謝辭…………………………………………………………………………i
中文摘要…………………………………………………………………...ii
英文摘要…………………………………………………………………..iii
目錄…………………………………………………………………….…..v
圖表目錄…………………………………………………………...……...ix
第一章、 文獻整理
1、 珊瑚的生殖……………………………………………………..…1
2、環境因子與珊瑚生殖的關係...........................................................2
3、生殖內分泌系統…………………………………………………….3
3-1脊椎動物的生殖內分泌系統………………………………….3
3-2無脊椎動物的生殖內分泌系統…………………………...…..5
4、生殖細胞的發育…………………………………………………...6
4-1脊椎動物生殖細胞的發育………………………...…………..7
4-2珊瑚生殖細胞的發育……………………………………...…..8
5、熱休克蛋白70(heat shock protein 70)…………………………….9
6、研究緣起與目的……………………………………………….…..12
第二章、基因選殖與及定量系統之建立……………………………….14
1、前言…………………………………………………………….…14
2、實驗物種………………………………………………………….15
3、實驗設計………………………………………………………….15
1.基因片段選殖………………………………………………......15
4、實驗方法………………………………………………………….15
1.全量核醣核酸(Total RNA) 之純化……………………………16
2.反轉錄-聚合?連鎖反應(Reverse Transcription - Polymerase Chain Reaction, RT-PCR)……………………………………...16
a. cDNA之合成………………………………………………..16
b.聚合?連鎖反應(PCR)………………………………………17
3.DNA萃取(DNA extraction)…………………………………….17
a.洋菜膠體之萃取法(gel extraction)…………………………..17
b. DNA重組接合(ligation) ……………………………………18
4.質體轉型(Transformation)……………………….…...………...18
5.微量質體DNA抽取(plasmid extraction)……………………...19
6.即時定量Real-time PCR系統之建立………………………….19
a.製備標準品RNA的模板…………………………………….19
b.標準品RNA製備…………………………………………….20
c.標準品單股cDNA (first strand cDNA)製備………………...21
d. Real-time quantification PCR……………………………….21
e.資料分析彙整………………………………………………..23
5、實驗藥品………………………………………………………….23
1.菌種、質體……………………………………………………....23
2.一般化學藥品…………………………………………………..23
3.酵素及生化分析試劑…………………………………………..24
4.實驗藥品及溶液配方……………..……………………………25
5.統計分析………………………………………………………..26
6、結果……………………………………………………………….27
1.珊瑚之heat shock protein 70a(hsp70a)與heat shock protein 70b(hsp70b)之基因選殖………………………………………...27
2.即時定量Real-time PCR系統之建立………………………….28
7、討論…………………………………….………………………….30
1. 珊瑚之hsp70a和hsp70b之基因選殖………………………...30
2.珊瑚基因標準品製備…………………………………………..30
第三章、繁殖季與非繁殖季珊瑚熱休克蛋白70基因表現之研究……45
1、前言………………………………………………………………...45
2、實驗生物…………………………………………………………...45
3、實驗設計…………………………………………………………...46
1.以半定量 PCR探討繁殖季和非繁殖季珊瑚基因的表現……46
2.以Real-time PCR探討繁殖季和非繁殖季珊瑚基因的表現…46
4、結果………………………………………………………………...46
1.探討繁殖季和非繁殖季珊瑚基因的表現……………………..46
5、討論………………………………………………………………...48
第四章、促性腺激素釋放素與珊瑚性賀爾蒙代謝之研究………….......53
1、前言………………………………………………………………...53
2、實驗物種…………………………………………………………...53
3、實驗設計…………………………………………………………54
1.以生殖腺細胞培養方式追蹤GnRH和「3H」Pregnenolone(「3H」P5)在生殖腺類固醇生成作用之機制………………………...54
4、實驗方法…………………………………………………………...54
1.以生殖腺細胞培養方式追蹤「3H」P5在生殖腺成熟過程之代謝產物…………………………………………………………….54
a.生殖腺組織添加「3H」P5 培養……………………………….54
b.類固醇激素萃取……………………………………………..55
c.高效能液相色層分析(High Performance Liquid Chromatogrphy, HPLC)………………………………….56
d.代謝物分析…………………………………………………..57
e.薄膜色層分析(Thin layer chromatography, TLC)………...57
f.類固醇二次分離之條件……………………………………...58
g.實驗藥品與溶液配方………………………………………..59
5、結果………………………………………………………………...59
6、討論………………………………………………………………...60
第五章、結論……………………………………………………………...73
參考文獻………………………………………………………………….75

圖表目錄
頁次
表1、選殖腎形真葉珊瑚之hsp70a胺基酸與其他動物之相似百分比…31
表2、選殖腎形真葉珊瑚之hsp70b胺基酸與其他動物之相似百分比…32
表3、雄性腎形珊瑚代謝產物經TLC鑑定後各類固醇之含量(dpm)…66
表4、雌性腎形珊瑚代謝產物經TLC鑑定後各類固醇之含量(dpm)…66
表5、雄性腎形珊瑚代謝產物經TLC鑑定後各類固醇之含量(dpm)…69
表6、雌性腎形珊瑚代謝產物經TLC鑑定後各類固醇之含量(dpm)…69
圖1、腎形真葉珊瑚hsp70a基因之片段核?酸及其演譯胺基酸序列…34
圖2、腎形真葉珊瑚hsp70a基因與其他物種基因之胺基酸序列比對…36
圖3、腎形真葉珊瑚hsp70b基因之片段核?酸及其演譯胺基酸序列…38
圖4、腎形真葉珊瑚hsp70b基因與其他物種基因之胺基酸序列比對…39
圖5、腎形真葉珊瑚 HSP70 與其他物種的HSP70做Neighbor joining 親緣性分析……………………………………………………….40
圖6、腎形真葉珊瑚hsp70a基因即時定量偵測系統(Real-time PCR)之建立…………………………………………………………….42
圖7、腎形真葉珊瑚hsp70b基因即時定量偵測系統(Real-time PCR)之建立…………………………………………………………….43
圖8、腎形真葉珊瑚β-actin基因即時定量偵測系統(Real-time PCR)之建立…………………………………………………………….44
圖9、腎形真葉珊瑚非繁殖季與繁殖季,雌雄珊瑚 hsp70a和hsp70b 基因表現量………………………………………………………….50
圖10、雌、雄腎形真葉珊瑚在不同月份hsp70a基因表現量的變化…51
圖11、雌、雄腎形真葉珊瑚在不同月份hsp70b基因表現量的變化…52
圖12、HPLC標準物分析圖譜……………………………………………63
圖13、繁殖季節追蹤珊瑚精巢組織代謝合成之激素種類……………64
圖14、繁殖季節追蹤珊瑚卵巢組織代謝合成之激素種類……………65
圖15、薄膜色層分析鑑定珊瑚生殖腺組織之類固醇激素(control組)..67
圖16、薄膜色層分析鑑定珊瑚生殖腺組織之類固醇激素(LHRH-A組)…………………………………………………………..……….68
圖17、圖13產物以TLC分離後各種類固醇激素濃度百分比之變化…69
圖18、圖14產物以TLC分離後各種類固醇激素濃度百分比之變化…70
圖19、推測腎形真葉珊瑚類固醇生合成之代謝路徑圖,虛線箭頭表示未進行偵測之代謝產物…………………………………………72
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