臺灣博碩士論文加值系統

本研究之目的在利用聚合酶連鎖反應-限制酶切割片段長度多態性(PCR-RFLP)與對偶基因特異性聚合酶連鎖反應(AS-PCR)針對山羊αs1酪蛋白進行基因分型，以得知台灣乳羊之αs1酪蛋白基因多態性，另進行羊乳酒精及熱安定性檢測，探討αs1酪蛋白基因多態性與羊乳酒精及熱安定性之關聯。採集160頭雜交乳用山羊之血液並萃取基因組DNA，利用PCR-RFLP與AS-PCR檢測αs1酪蛋白基因型，可得到下列11種基因型：AA、AE、AF、A01、EE、EF、EN、E01、FF、F01及N01，基因型頻率分別為0.13、0.13、0.29、0.02、0.02、0.19、0.01、0.03、0.13、0.04及0.01，以AF基因型出現之頻率最高(0.29)，N01基因型之頻率最低(0.01)；對偶基因則以αs1酪蛋白F對偶基因出現之頻率最高(0.40)，顯示台灣雜交乳用山羊普遍帶有αs1酪蛋白F對偶基因。αs1酪蛋白AF、EF、FF及F01基因型之羊乳蛋白質經SDS-PAGE可得到大小約23 kDa之蛋白質特異片段，顯示此片段可做為SDS-PAGE篩選αs1酪蛋白F對偶基因之特異蛋白質。另採集正值泌乳期之74頭經產及26頭初產母羊，總計100頭雜交山羊之羊乳，分別進行酒精(35-65%)及熱安定性(90-150°C)檢測發現，平均酒精安定性為47.7±5.1%，平均熱安定性則為108.2±4.9°C，兩者具有正相關(p<0.05, r=0.29658)，顯示酒精安定性可做為羊乳熱安定性之依據。經產及初產母羊方面，初產母羊之羊乳酒精及熱安定性皆極顯著低於經產母羊(p<0.001)，顯示分娩胎次可能會影響羊乳酒精及熱安定性，其影響機制仍有待後續研究。αs1酪蛋白基因型方面，不同αs1酪蛋白基因型之羊乳酒精及熱安定性雖未達顯著水準(p>0.05)，但帶有F對偶基因之αs1酪蛋白基因型(AF、EF及FF基因型)具有較高之羊乳酒精及熱安定性，αs1酪蛋白F對偶基因可作為熱安定性乳羊之篩選基因。

The purpose of this study is to investigate the αs1-casein gene polymorphisms in Taiwan dairy goats were analyzed by polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) and allele-specific polymerase chain reaction (AS-PCR). The ethanol and heat stability of goat milk were also detected to investigate the relationship with αs1-casein genotype. Genomic DNA isolated in blood samples from 160 dairy goats was detected for genotypes of αs1-casein gene (CSN1S1) by PCR-RFLP and AS-PCR. Eleven αs1-casein genotypes were found: AA, AE, AF, A01, EE, EF, EN, E01, FF, F01 and N01. The frequencies of these genotypes were 0.13, 0.13, 0.29, 0.02, 0.02, 0.19, 0.01, 0.03, 0.13, 0.04 and 0.01, respectively. The most common genotype was AF (0.29), and the least common was N01 (0.01). In CSN1S1 alleles, the frequency of F allele (0.40) was the highest than others, which showed that CSN1S1 F allele was the most common allele of all examined dairy goats. Since SDS-PAGE revealed that the milk of AF, EF, FF and F01 genotypes had a specific protein fragment at about 23 kDa, the fragment could become a specific protein for selecting CSN1S1 F. Milk collected from 100 milking dairy goats, including 74 does and 25 maiden does, for detection of ethanol and heat stability revealed ethanol concentrations of ranging from 35 to 65% and temperatures of ranging from 90 to 140°C. The average ethanol and heat stability of milks were 47.7±5.1% and 108.2±4.9°C, respectively. Ethanol stability also correlated with heat stability (p&lt;0.05, r=0.29658), , so ethanol stability is in accordance with heat stability of goat milk. The ethanol and heat stability in maiden does were both significantly lower than in does (p&lt;0.001), which showed that parity may affect the ethanol and heat stability of goat milk. However, the associated mechanisms require further study. In αs1-casein genotype, the ethanol and heat stability had no significantly difference between different αs1-casein genotypes (p>0.05), but the ethanol and heat stability of the genotype with F allele (AF, EF and FF genotype) were both higher than others. These comparison results showed that CSN1S1 F could be a selected gene for dairy goat of heat stability.

目錄
頁次
中文摘要…………………………………………………………...…....II
英文摘要…………………………………………………………...…...III
圖次………………………………………………………………...…VIII
表次…………………………………………………………………...IX
前言……………………………………………………………1
壹、文獻探討..............................................................................................3
　一、乳中酪蛋白之合成與特性..............................................................3
(一) 酪蛋白之合成.....................................................................3
(二) 酪蛋白之特性.....................................................................3
　二、酪蛋白膠粒結構之模型..................................................................4
　　　(一) Coat-core models................................................................4
(二) 次膠粒／次單元模型.........................................................5
(三) 內部結構模型.....................................................................5
　三、羊乳酪蛋白膠粒..............................................................................6
四、山羊αs1酪蛋白基因多態性.............................................................8
(一) αs1酪蛋白A、B及C對偶基因(高表現量) .........................8
(二) αs1酪蛋白E對偶基因(中表現量) ....................................10
(三) αs1酪蛋白D及F對偶基因(低表現量) ............................10
(四) αs1酪蛋白01及N對偶基因(無表現量) .........................10
五、山羊αs1酪蛋白基因多態性檢測.................................................11
(一) 限制酶片段長度多態性....................................................11
(二) 對偶基因特異性聚合酶連鎖反應...................................12
(三) 其他方法………………...................................................12
六、羊乳熱安定性……......................................................................13
(一) αs1酪蛋白基因多態性及αs1酪蛋白含量...........................14
(二) pH值..................................................................................15
(三) 磷酸鹽、酪蛋白及乳清蛋白含量.....................................15
(四) 生乳保存方式...................................................................16
七、羊乳酒精安定性…......................................................................16
貳、材料與方法……................................................................................18
一、山羊αs1酪蛋白基因型及表現型………….…............................18
(一) 試驗樣本之採集………...................................................18
(二) 基因組DNA萃取及定量……………….......................18
(三) αs1酪蛋白基因分型…………………...............................22
(四) 統計分析….………..........................................................31
(五) SDS-PAGE……………….................................................32
二、羊乳酒精及熱安定性..................................................................35
(一) 試驗樣品之採集………….………................................37
(二) 羊乳酒精安定性…………….…………........................37
(三) 羊乳熱安定性……………...........................................37
(四) 統計分析….………….……...........................................37
参、結果與討論........................................................................................39
一、山羊αs1酪蛋白基因多態性……….............................................39
(一) PCR-RFLP……….…......................................................39
(二) AS-PCR……….…..........................................................43
(三) αs1酪蛋白基因型……….…............................................49
(四) αs1酪蛋白基因頻率.........................................................51
(五) 蛋白質電泳………….....................................................55
二、羊乳酒精及熱安定性……..........................................................57
(一) 羊乳酒精及熱安定性之相關性……….…....................57
(二) 分娩胎次與羊乳酒精及熱安定性之關聯.....................58
(三) αs1酪蛋白基因型與羊乳酒精及熱安定性之關聯.........58
肆、結論....................................................................................................64
參考文獻………………………………………………….……………65




圖次
頁次
圖 1 山羊αs1酪蛋白變異之演化…………………...…………...….....7
圖 2 山羊αs1酪蛋白基因型與表現型檢測之試驗流程…….............19
圖 3 檢測山羊αs1酪蛋白對偶基因之示意圖…...……………...…...24
圖 4 山羊αs1酪蛋白A對偶基因之部分序列……………….............25
圖 5 山羊αs1酪蛋白A對偶基因之部分序列………….…...............28
圖 6 山羊αs1酪蛋白對偶基因第12個介入子之序列及引子位置....30
圖 7 SDS-PAGE分析之試驗流程………………………...................33
圖 8 羊乳酒精及熱安定性測定之試驗流程……….…………..…...36
圖 9 利用PCR-RFLP檢測山羊αs1酪蛋白對偶基因之示意圖..…....40
圖 10 山羊αs1酪蛋白基因經由PCR增殖所得之片段以XmnI限制酶切割之產物電泳圖………………………………...………......42
圖 11 利用AS-PCR檢測山羊αs1酪蛋白對偶基因之示意圖..…........44
圖 12 利用αs1 19F及αs1 19R引子針對山羊基因組DNA增殖αs1酪蛋白對偶基因之PCR產物電泳圖………………………..….......46
圖 13 利用αs1 12F、αs1 12R及αs1 01引子針對山羊基因組DNA增殖αs1酪蛋白對偶基因之PCR產物電泳圖………..…………......48
圖 14 不同αs1酪蛋白基因型之羊乳SDS-PAGE…..……....................56

表次
頁次
表 1 山羊αs1酪蛋白變異/對偶基因之差異…………………..…........9
表 2 山羊αs1酪蛋白基因分型之檢測方法………....………...……..23
表 3 山羊αs1酪蛋白基因型之PCR產物與限制酶切割片段長度....50
表 4 160頭雜交泌乳山羊之αs1酪蛋白基因型頻率………..…....…52
表 5 160頭雜交泌乳山羊之αs1酪蛋白對偶基因頻率…………..…53
表 6 比較不同文獻之αs1酪蛋白對偶基因頻率結果………….....…54
表 7 初產及經產母羊羊乳酒精及熱安定性之一般線性分析結果..59
表8 不同αs1酪蛋白基因型羊乳酒精及熱安定性之一般線性分析結
果………………….………………………...….………….……62

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