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研究生:薛丁瑋
研究生(外文):HSUEH, TING-WEI
論文名稱:乳山羊αs1-酪蛋白基因限制片段長度多態性與產乳特性之關係
論文名稱(外文):The relationship between milk characteristics and RFLPs found in αs1-casein gene of dairy goats
指導教授:鄭 登 貴姜 延 年
指導教授(外文):Teng- Kuei ChengYan- Nian Jiang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:畜產學研究所
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:2000
畢業學年度:88
語文別:中文
論文頁數:102
中文關鍵詞:產乳特性多態性
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根據以往研究顯示山羊乳中s1-酪蛋白具有多種遺傳變異型,且此等變異型與乳生產性狀關係密切。本研究之目的,在於應用限制酶截切片段長度多態性(restriction fragment length polymorphism, RFLP)分析技術,試圖從DNA層次區分出不同品種乳山羊之s1-酪蛋白基因型的多態性,並探討此等多態性與乳成分及乳熱安定性等之關係,冀能建立一簡便且可靠之遺傳標記(genetic marker),俾可提供未來據以進行乳山羊之選拔,從而改善其羊乳之生產特性。
本試驗共計使用源自台大與民間乳羊場飼養之阿爾卑因羊51頭、薩能羊33頭與雜交種羊(阿爾卑因×奴比亞)42頭,分別採集其乳樣與血樣進行分析。採集之乳樣,除分析乳蛋白質、乳脂肪、乳糖、總固形物等一般乳成分外,亦測定乳中之體細胞數及乳對加熱導致凝集之耐受性。採集之血樣分別經萃取其基因組DNA後,再使用AvaI、BamHI、BglI、EcoRI、EcoRV、HindIII、NcoI、PvuII、SacI與TaqI等十種不同核酸限制酶分別進行截切,並以s1-酪蛋白基因前端第四表現子至第七表現子區域長度約1.75 kb之DNA作為探針,進行南方氏雜合( Southern hybrodization)分析,俾確認各羊隻之αs1-酪蛋白基因的RFLP;並配合前述乳樣之各項測試結果,進行相關性分析。
試驗結果顯示,前述測試之十種核酸限制酶中,僅EcoRI與TaqI兩者對乳山羊αs1-酪蛋白基因可截切出RFLP;其餘限制酶截切結果,則均無RFLP可言。根據TaqI截切所獲得之DNA片段長度顯示,αs1-酪蛋白基因具有三種對偶基因,其長度分別為A型16.5 kb、B型9.0 kb與C型5.5 kb/3.0 kb。經EcoRI截切者,則可獲得兩種對偶基因,其DNA片段長度分別為M型4.0 kb及N型1.5 kb。將前述乳山羊αs1-酪蛋白基因的RFLP與山羊乳之生產性狀比較結果顯示,就TaqI截切所產生之多態性而言,在乳脂肪含量(AC>AB,BB)、乳蛋白質含量(BC> AA,BB,AB) 、乳糖含量(BC 最高)及總固形物含量(AC,BC>AA,AB,BB)等,各RFLP型別間均具有顯著差異(P<0.05);惟各基因型與體細胞數及乳加熱凝集性二者,則均無顯著差異(P>0.05)。就EcoRI截切所產生之RFLP而言,則與前述山羊乳各個性狀間之關係性,均未達統計上之顯著差異性( P>0.05);惟試驗數據亦顯示,前述山羊乳之各個性狀,顯著受到場別、品種與胎次等之影響。
此外,針對乳山羊αs1-酪蛋白cDNA序列設計之引子,擴增所獲得之包含啟動子區域至第十七表現子片段,經使用前述不同之核酸限制酶進行截切後,證明AseI限制酶對αs1-酪蛋白基因之上游啟動子區域(promoter region)確實呈現有RFLP。進一步針對該特定區域之基因片段進行定序分析比較結果,更證實乳山羊αs1-酪蛋白基因之上游啟動子序列,於-396 bp處呈現有G/A點之突變現象,遂導致該區域經AseI限制酶截切結果,產生P與Q者兩種不同型別之RFLP;就該二型與前述山羊乳各性狀之關係而言,羊隻之為P型者,顯示其乳脂肪、乳蛋白質、乳糖與總固形物含量等,均顯著高於Q型羊隻者(P<0.05)。鑑於一般基因之啟動子區域,常含有若干特定之轉錄因子辨識序列,因此推測αs1-酪蛋白基因之啟動子發生前述點突變區,可能對其轉錄作用亦有影響,惟其可能涉及之轉錄因子及詳細之作用機制,則尚待進一步深入探討,始克有明。
Purposes of the present study were to investigate whether the restriction fragment length polymorphism (RFLPs) in s1-casein gene of the dairy goats appeared to be correlated to their milk characteristics including the milk composition and the milk heat stability. It is anticipated that a reliable and applicable genetic marker based on the molecular level of RFLPs found in goat s1-casein gene could be easily fitted to future breeding scheme for selection those goats with better milk characteristics in both of the composition and the heat stability. To achieve the above purposes, Milk and blood samples taken from a total of 126 goats, including 51 alpine, 33 saanen and 42 Alpine×Nubian hybrid from herds of either animal farm of National Taiwan University or several local-farms in southern parts of Taiwan, were subjected to assessments of the milk characteristics and the RFLP analysis, respectively, and the data obtained from each parameter were subjected to statistical analysis of their correlation. Assessments of milk characteristics included that of fat, protein, lactose, total solid portion, somatic cell counts contained in each of goat milk sample and the heat stability of the milk, i.e. the time required for causing goat milk appeared to be heat-coagulated at 138 oC. For RFLP analysis, genomic DNA extracted from each of blood samples had been digested with the restriction endonuclease before they were subjected to southern-blot analysis by the use of a 1.75 kb gDNA fragment, located between exons 4-7 of the caprine s1-casein gene, as the probe for hybridization. Experimental results showed that there was virtually no RFLP existed in the goat s1-casein gene when their gDNA had been digested with the following eight restriction enzymes including AvaI, BamHI, BglI, EcoRV, HindIII, NcoI, PvuII and SacI etc. However, various of RFLPs were evidenced by the use of TaqI which resulted in a total of six genotypes (AA、AB、BB、BC、CC、AC) and the length of fragments A, B and C allele were identified to be 16.5, 9.0, and 5.5/3.0 kb, respectively. Similarly, EcoR I produced three genotypes (MM、MN and NN) and the fragment M and N allele were 4.0 kb and 1.5 kb, respectively. The least squares analysis showed possible associations between s1-casein genotype and milk composition. Genotype obtained by Taq I digestion had significant differences in fat content (AC>AB and BB), milk protein content (BC>AA,BB, and AB), lactose (BC>other genotypes) and milk total solids (AC and BC>AA, AB, and BB)(P<0.05), but there were no significant relationships observed between genotypes and somatic cell counts or heat stability (P>0.05). In addition, no significant relationships were observed between genotypes obtained by EcoR I digestion and milk characteristics. Furthermore, significant differences were noted in milk composition among breeds and herds.
In the further experiment, six pairs of primer deduced from exons of the published goat s1-casein cDNA sequences were used for PCR(polymerase chain reaction) amplification of fragments between promoter region and exon 17. When PCR products were digested with various restriction enzymes, different fragment length polymorphisms were obtained by Ase I endonuclease. The DNA autosequencing results showed a point mutation at position -396 in the 5'flanking region of s1-casein gene that had separate into two types( P and Q), it had significant differences in milk composition. It is likely that the point mutation sequence may provide some transcription factor binding sites involved in the regulation of the s1-casein gene expression, thereby resulting in a difference in milk composition.
摘要……………………………………………………………1
緒言……………………………………………………………3
文獻檢討………………………………………………………4
材料與方法……………………………………………………37
結果與討論……………………………………………………52
結論……………………………………………………………82
參考文獻………………………………………………………83
英文摘要………………………………………………………95
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