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研究生:黃章文
研究生(外文):Chang-Wen Huang
論文名稱:褐色菜鴨基因組、粒線體DNA與血清蛋白多態性之研究
論文名稱(外文):Studies on the Polymorphisms of Genomic, Mitochondrial DNA and Serum Proteins in Brown Tsaiya Ducks
指導教授:鄔維雅黃木秋黃木秋引用關係
指導教授(外文):Roger RouvierMu-Chiou Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:畜產學系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:121
中文關鍵詞:褐色菜鴨基因組DNA粒線體DNA血清蛋白多態性
外文關鍵詞:Brown Tsaiya duckGenomic DNAMitochondrial DNASerum ProteinPolymorphism
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本試驗目的是利用分子遺傳標記探討褐色菜鴨基因組、粒線體DNA與血清蛋白之多態性。以增殖片段長度多態性(amplified fragment length polymorphisms, AFLP)、逢機增殖多態性DNA(random amplified polymorphic DNA;RAPD)、逢機增殖微衛星多態性(random amplified micorsatellite polymorphisms;RAMPO)等DNA指紋技術、單股構型多態性(single-strand comformation polymorphism;SSCP)、序列分析、Titan膠體以及PAGE等電泳,分析於選拔受精持續試驗中之選拔與對照兩系群褐色菜鴨基因組、粒線體DNA D-loop區域及血清蛋白之多態性。利用EcoRI/TaqI 16種引子組合增殖出AFLP態樣之結果顯示,兩系群菜鴨皆可產生多態性環帶。依據30種逢機引子所增殖出之RAPD態樣,並估算選拔系群菜鴨所得之平均共有環帶出現頻率之範圍為0.69 ± 0.20∼0.97 ± 0.06,平均為0.85 ± 0.1;對照系群則為0.68 ± 0.21∼0.98 ± 0.25,平均為0.82 ± 0.1,即選拔系菜鴨之遺傳純度較高於對照系,但差異不顯著(P>0.05);兩系群之遺傳距離及相似性分別為0.15及88.57%。RAMPO指紋則可進一步呈現於RAPD指紋中無法顯現之環帶態樣。在粒線體DNA D-loop區域393-bp片段之SSCP分析結果發現,所有供試菜鴨均只呈現單一種態樣,經由序列分析之比對結果,兩系群菜鴨共發現3個同類置換之核酸變異,序列分歧度為0.42%。另以Titan及PAGE分析兩系群菜鴨之血清蛋白質成分及特定蛋白型之結果發現,α2球蛋白之比例以選拔系群較對照系群之鴨隻為高,於兩系群間有顯著差異(P<0.05)。而鴨隻血清特定蛋白型之分析結果,發現兩系群菜鴨血清中之前白蛋白、白蛋白和鐵合蛋白具有多態性,且於Tf多態性表型及基因頻度之分佈,以雜合子個體之出現頻度較純合子者高。其中所出現之不同環帶是否與鴨隻重要經濟性狀相關,又是否可做為分子遺傳標記,提供菜鴨早期選汰之指標,尚待進一步的探討。

The purpose of this study was to use molecular genetic markers to explore the polymorphism of genomic, mitochondrial DNA and serum proteins in Brown Tsaiya ducks which were a selected and control line from selection experiment to increase the duration of fertility. The techniques: AFLP, RAPD, RAMPO, SSCP, sequencing, TITAN gel and PAGE electrophoresis were used as the tools in these studies. The results of AFLP patterns of Brown Tsaiya ducks from Selected and Control lines used sixteen EcoRI/TaqI primer combinations. Polymorphic bands were identified. Thirty arbitrary primers were used to amplify genomic DNA of Brown Tsaiya ducks. The results showed that the average band sharing frequency of RAPD fingerprints in Tsaiya ducks was 0.85±0.1(0.69 ±0.20∼0.97±0.06)and 0.82±0.1(0.68 ±0.21∼0.98±0.25)in Selected and Control line, respectively. They indicated a higher homozygosity in the Selected line than in the Control line, but the difference between the two lines was not significant(P>0.05). The genetic distance and similarity were 0.15 and 88.57%. The same site of band appeared in RAPD fingerprints but sometime the band disappeared in RAMPO fingerprints. It indicated that the band of the same site in RAPD fingerprints had DNA sequence probably different from the band of RAMPO fingerprints. There were only the same SSCP type in the D-loop region of mtDNA in Tsaiya ducks. In sequencing results showed that 3 nucleotide variant of the 393-bp fragment in mtDNA D-loop region were found in this study. They all were transitions. According to the nucleotide variances to estimate the diversities between the two lines of Brown Tsaiya ducks was above 0.42%. The results of Titan gel serum proteins analysis showed that percentages ofα2-globulin between lines of Brown Tsaiya ducks differ significantly(P<0.01). Using PAGE to analyze the patterns showed that the pre-albumin, albumin and transferrin in Selected and Control lines presented polymorphisms. The Tf phenotype and frequency of heterozygotes was much higher than that of homozygotes. So, preliminary results showed that the band patterns of AFLP, RAPD, TITAN gel or PAGE electrophoresis presented polymorphisms. To know whether some bands could be related to the performance of Tsaiya duck production will need to do further studies in the future experiments.

壹、中文摘要…………………………………………………………………………1
貳、前言………………………………………………………………………………3
參、文獻檢討…………………………………………………………………………4
一、台灣菜鴨養育概況………………………………………………………………4
(一)養鴨事業之發展及現況………………………………………………………4
(二)台灣地區現有鴨隻品種………………………………………………………5
(三)菜鴨受精持續性之遺傳改良…………………………………………………6
(四)菜鴨生產事業在台灣的重要性………………………………………………8
二、家畜禽數量性狀之改良…………………………………………………………9
(一)家畜禽重要經濟性狀…………………………………………………………9
(二)傳統數量性狀之遺傳改良……………………………………………………10
(三)數量性狀基因座………………………………………………………………11
三、基因組DNA多態性及其應用……………………………………………………12
(一)DNA遺傳標記之分類…………………………………………………………12
(二)測定基因組DNA多態性之方法………………………………………………15
1.限制片段長度多態性………………………………………………………………15
2.迷你衛星多態性……………………………………………………………………15
3.微衛星多態性.………………………………………………………………………16
4.逢機增殖多態性DNA………………………………………………………………17
5.增殖片段長度多態性………………………………………………………………17
(三)遺傳標記應用於數量性狀基因座基因圖譜之定位…………………………21
四、粒線體DNA多態性及其應用……………………………………………………23
(一)粒線體DNA之遺傳特性………………………………………………………23
(二)SSCP分析原理及發展…………………………………………………………25
(三)粒線體DNA與家畜重要經濟性狀……………………………………………26
五、血清蛋白多態性與數量性狀……………………………………………………27
(一)血清蛋白之種類及其功能……………………………………………………27
(二)血清蛋白及血型多態性之應用………………………………………………28
(三)血液蛋白多態性為數量遺傳標記……………………………………………29
肆、試驗部分…………………………………………………………………………30
試驗一. 褐色菜鴨基因組DNA之AFLP指紋分析……………………………………31
一、摘要………………………………………………………………………………31
二、前言………………………………………………………………………………31
三、材料與方法………………………………………………………………………32
(一)試驗動物與樣品處理…………………………………………………………32
(二)基因組DNA之萃取……………………………………………………………32
(三)AFLP指紋技術之建立…………………………………………………………34
四、結果與討論………………………………………………………………………39
(一)褐色菜鴨基因組DNA之限制切割與連接作用……………………………39
(二)篩選性引子增殖出多態性環帶………………………………………………40
五、結論………………………………………………………………………………40
六、英文摘要…………………………………………………………………………48
試驗二. 褐色菜鴨基因組DNA之RAPD及RAMPO指紋分析………………………49
一、摘要………………………………………………………………………………49
二、前言………………………………………………………………………………49
三、材料與方法………………………………………………………………………50
(一)試驗動物與樣品處理…………………………………………………………50
(二)基因組DNA之萃取……………………………………………………………50
(三)RAPD指紋技術之建立..………………………………………………………52
(四)RAMPO指紋技術之建立………………………………………………………53
(五)RAPD指紋技術之分析………………………………………………………55
四、結果與討論………………………………………………………………………56
(一)逢機增殖多態性DNA指紋…………………………………………………… 56
(二)逢機增殖微衛星多態性指紋…………………………………………………58
五、結論………………………………………………………………………………59
六、英文摘要…………………………………………………………………………66
試驗三. 以Tsaimt P1, P2之PCR分析褐色菜鴨粒線體DNA D-loop區域SSCP及其
序列分析……………………………………………………………………68
一、摘要………………………………………………………………………………68
二、前言………………………………………………………………………………68
三、材料與方法………………………………………………………………………69
(一)試驗動物與樣品處理…………………………………………………………69
(二)基因組DNA之萃取……………………………………………………………69
(三)粒線體DNA D-loop區域SSCP分析……………………………………………71
(四)粒線體DNA D-loop區域序列分析……………………………………………73
四、結果與討論………………………………………………………………………77
(一)粒線體DNA D-loop區域SSCP分析……………………………………………77
(二)粒線體DNA D-loop區域序列分析……………………………………………77
五、結論………………………………………………………………………………78
六、英文摘要…………………………………………………………………………84
試驗四. 褐色菜鴨血清蛋白質多態性分析…………………………………………85
一、摘要………………………………………………………………………………85
二、前言………………………………………………………………………………85
三、材料與方法………………………………………………………………………86
(一)試驗動物………………………………………………………………………86
(二)血樣採集與血清分離…………………………………………………………86
(三)醋酸纖維膠片電泳分析血清蛋白質…………………………………………87
(四)聚丙烯醯胺膠體電泳分析血清蛋白型………………………………………89
(五)統計分析………………………………………………………………………91
四、結果與討論………………………………………………………………………92
(一)醋酸纖維膠片電泳分析血清蛋白質…………………………………………92
(二)聚丙烯醯胺膠體電泳分析血清蛋白型………………………………………96
五、結論………………………………………………………………………………97
六、英文摘要…………………………………………………………………………101
伍、總結………………………………………………………………………………102
陸、參考文獻…………………………………………………………………………104
柒、英文摘要…………………………………………………………………………120

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