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研究生:梁筱梅
研究生(外文):Liang, Hsiao-Mei
論文名稱:熱休克蛋白70基因多態型對台灣土雞急性熱緊迫反應、生長與產蛋性能之關聯性
論文名稱(外文):Association of heat shock protein 70 gene polymorphisms with acute thermal stress, growth, and egg production of Taiwan native chicken
指導教授:許岩得洪國翔洪國翔引用關係
指導教授(外文):Hsuuw, Yan-DerHung, Kuo-Hsiang
口試委員:邱文石夏良宙鄭裕信李嘉偉
口試日期:2017-05-13
學位類別:博士
校院名稱:國立屏東科技大學
系所名稱:生物資源研究所
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:2017
畢業學年度:105
語文別:中文
論文頁數:105
中文關鍵詞:熱休克蛋白70基因熱緊迫台灣土雞
外文關鍵詞:Heat shock protein 70 geneheat stressTaiwan native chicken
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高溫氣候對於雞隻生長與生產相當不利,因此選育耐熱品系雞隻甚為重要。本試驗以聚合脢連鎖反應-單鏈構象多態性(Polymerase chain reaction- single strand conformation polymorphism, PCR-SSCP)方法鑑定HSP70基因型之多態型,評估基因多態型對雞隻急性熱緊迫反應,並分析HSP70基因多態型對雞隻生長及產蛋性能之影響,期作為育成耐熱品系雞隻之參考。308隻近親品系臺畜一號L9品系雞隻於12週齡採血分析基因型,鑑定HSP70基因258位點上鹼基A→G轉換,產生A258A、A258G及G258G等3種基因型。雞群依基因型分組進行急性耐熱性試驗。每組雞隻分別置於40、42、45℃環境溫度達1至2小時,分析呼吸頻率、洩殖腔溫度、血漿CK、T3及腿肉mRNA表現量以評估急性熱緊迫之生理反應。試驗期間紀錄雞隻出生重、16週齡體重、產蛋初產日齡、初產蛋重、初產體重及至40週齡產蛋數以分析HSP70基因多態型對雞隻生長及產蛋性能影響。試驗結果雞隻於40℃達1小時,其呼吸頻率、洩殖腔溫度、血漿CK活性及T3濃度等生理變化於各基因組間均無顯著差異;雞隻於40℃達2小時,其呼吸頻率、血漿CK活性及T3濃度等生理變化於各基因組間均無顯著差異,而洩殖腔溫度以G258G基因型雞隻顯著最低;雞隻於42℃達1小時,呼吸頻率增加程度以A258G基因型雞隻顯著最高,且G258G基因型雞隻顯著最低(P=0.008),而洩殖腔溫度因A258A及A258G基因型雞隻已超過測量範圍(即44℃),因此未予後續分析;血漿CK活性及T3濃度等生理變化於各組間均無顯著差異;當雞隻於42℃達2小時,呼吸頻率增加程度仍以A258G基因型雞隻顯著最高,G258G基因型雞隻顯著最低(P=0.0026);而血漿CK活性增加程度以A258G基因型雞隻顯著最高,A258A基因型雞隻顯著最低(P=0.025);血漿T3濃度則以A258A基因型雞隻降低程度最大,G258G基因型雞隻降低程度顯著最小(P=0.035);腿肉mRNA表現量以A258A基因型最高,G258G基因型顯著最低。雞隻於45℃環境溫度達1小時,其腿肉mRNA表現量亦以A258A基因型最高,G258G基因型顯著最低,然而雞隻開始有死亡情形發生,顯示45℃為近親品系臺畜一號L9品系之致死溫度。在生長性能與產蛋性能試驗結果顯示,公雞與母雞16週齡增重均以A258A基因型雞隻顯著最高,G258G基因型雞隻顯著最低(P=0.041與P <0.001),初產蛋重及40週齡產蛋數於各組間均無顯著差異。試驗顯示HSP70基因多態型與雞隻耐熱能力之相關性仍需進一步研究,然而以HSP70基因作為選育目標,對雞隻產蛋性能之表現並無不良之影響。
Heat stress is one of the most challenging environmental conditions affecting commercial poultry. It severely affects growth and egg production. Therefore, breeding thermal-tolerance chickens is important for poultry. The 308 Taishu no. 9 LRI chickens were used to examine the genotypes of the HSP70 gene performed by Polymerase chain reaction- single strand conformation polymorphism (PCR-SSCP) analysis. The polymorphisms identified were A258A, A258G, and G258G. Twenty 12-week old birds were randomly selected from each genotype and exposed to 40℃, 42℃ and 45℃ ambient temperature for 1 and 2h to evaluate acute thermal tolerance. The physiological responses were examined among geontypes, including respiratory rates, cloacal temperature, enzymatic activity of creatine kinase (CK), triiodothyronine (T3) levels, and HSP70 mRNA expression of leg meat. The body weight at hatching, body weight at 16 weeks of age, age at first egg, egg weight at first egg, body weight at first egg and the number of eggs laid up to 40 weeks of age were recorded to evaluate the association of these genotypes with growth and egg production. Results showed that there were no significant differences in respiratory rates, cloacal temperature, enzymatic activity of CK and T3 levels among the genotypes after 1 h at 40℃. After 2 h at 40℃, there were no significant differences in respiratory rates, CK activity and T3 levels among the genotypes. The G258G genotype displayed lower cloacal temperature. After 1 h at 42℃, the A258G genotype displayed the highest respiratory rates, and G258G genotype displayed lowest respiratory rates among the genotypes (P=0. 008). The cloacal temperatures of the A258A and A258G genotypes exceeded the reading upper range (44℃) of the thermometer, however, the experiments not to be processed. Under 42℃ for 2h, the A258G genotype had significantly the highest respiratory rate and G258G genotype had lowerest; A258G genotype also exhibited highest level of CK activity, and A258A genotype had lowerest; A258A genotype exhibited significantly highest droping level of plasma T3, and G258G genotype had lowerest. However, chickens at 42℃for 1h began to die, and 45 ℃ is the lethal temperature for Taishu no. 9 LRI chickens. For growth and egg production trials, the body weight gain at 0-16 weeks of age differed among the genotypes for males (P=0. 041) and females (P<0.001), the A258A genotype exhibited the greatest weight gain at 0-16 weeks of age for both genders, and G258G had the lowest. There were no significant differences among genotypes regarding egg weight at first egg or the number of eggs laid until 40 weeks of age. Further researches are needed for association of heat shock protein 70 gene polymorphisms with acute thermal stress in chickens. Selection heat shock protein 70 gene in chickens for resistance to heat stress had no negative effects on egg production.
摘要------------------------------------------- I
謝誌------------------------------------------- V
目錄------------------------------------------- VII
圖表目錄---------------------------------------- IX
壹、前言---------------------------------------- 1
貳、文獻探討------------------------------------ 2
一、熱緊迫對雞隻影響-------------------------- 2
(一) 緊迫的定義---------------------------- 2
(二) 緊迫程度------------------------------ 2
(三) 緊迫反應------------------------------ 2
(四) 熱緊迫對雞隻生長和產蛋性能的影響-------- 5
(五) 評估雞隻熱緊迫的生理因子--------------- 9
二、熱休克蛋白質------------------------------ 16
(一) 熱休克蛋白質的發現--------------------- 16
(二) 熱休克蛋白質的分類--------------------- 19
(三) 熱休克蛋白質70的結構------------------- 19
(四) 熱休克蛋白質70的合成------------------- 23
(五) 熱休克蛋白質70的功能------------------- 23
(六) 熱休克蛋白質70應用於雞隻耐熱性的研究---- 24
参、材料與方法---------------------------------- 27
一、試驗設計---------------------------------- 27
二、試驗動物---------------------------------- 27
三、雞隻管理---------------------------------- 27
(一) 雞隻週齡之資料收集-------------------- 27
(二) 餵飼---------------------------------- 28
(三) 飼養溫度與濕度------------------------ 28
(四) 防疫計畫------------------------------ 28
四、基因型鑑定-------------------------------- 31
(一) 萃取DNA------------------------------- 31
(二) 聚合酶鏈鎖反應(Polymerase chain reaction,
PCR)----------------------------------31
(三) SSCP--------------------------------- 32
五、急性耐熱性試驗----------------------------- 32
(一) 急性熱處理---------------------------- 32
(二) 分析項目------------------------------ 32
六、統計分析---------------------------------- 35
肆、結果---------------------------------------- 36
一、基因型鑑定-------------------------------- 36
二、HSP70基因多態型對急性熱緊迫雞隻之耐熱性試驗---39
(一) 熱處理溫度及時間對雞隻之呼吸頻率、CK活性、T3
及HSP70 mRNA變化之影響---------------- 39
(二) HSP70基因型對熱緊迫雞隻呼吸頻率、CK活性、T3
及HSP70 mRNA變化之影響---------------- 48
三、HSP70基因多態型對生長性能之影響------------- 65
四、HSP70基因多態型對產蛋性能之影響------------- 65
伍、討論---------------------------------------- 68
一、基因型鑑定-------------------------------- 68
(一) 檢測HSP70基因之SNP-------------------- 68
(二) 結語---------------------------------- 70
二、HSP70基因多態型對急性熱緊迫雞隻生理反應之影響-71
(一) 呼吸頻率------------------------------ 71
(二) 體溫---------------------------------- 72
(三) 血漿CK活性---------------------------- 73
(四) 血漿T3濃度---------------------------- 75
(五) HSP70 mRNA表現量---------------------- 76
(六) 結語---------------------------------- 78
三、HSP70基因多態型對經濟性狀之影響------------- 79
(一) 對生長性能之影響----------------------- 79
(二) HSP70基因多態型對產蛋性能之影響--------- 79
(三) 結語--------------------------------- 80
陸、結論---------------------------------------- 81
柒、參考文獻------------------------------------ 82
捌、作者簡介------------------------------------ 101


圖 表 目 錄
表次
表1. 熱休克/緊迫蛋白的分類----------------------- 20
表2. 雞隻各階段餵飼之飼糧組成-------------------- 29
表3. 雞隻防疫計畫------------------------------- 30
表4. 熱處理溫度與時間對雞隻呼吸頻率、CK活性、T3濃度
與HSP70 mRNA表現量之影響------------------- 40
表5. 雞隻熱緊迫之呼吸頻率、CK 、T3及mRNA變化之相關
性分析------------------------------------ 45
表6. 熱處理溫度及時間對呼吸頻率、CK、T3及mRNA表現量
變化之交互作用----------------------------- 47
表7. HSP70基因型對熱緊迫雞隻呼吸頻率之影響-------- 49
表8. HSP70基因型對熱緊迫雞隻洩殖腔溫度之影響------ 52
表9. HSP70基因型對熱緊迫雞隻血漿CK活性之影響------ 54
表10. HSP70基因型對熱緊迫雞隻血漿T3濃度之影響----- 57
表11. HSP70基因型對熱緊迫雞隻腿肉HSP70 mRNA表現量
之影響------------------------------------ 60
表12. 熱緊迫下HSP70基因型、溫度及時間因子對呼吸頻率
、CK及T3之交互作用------------------------ 63
表13. HSP70基因多態型對生長性能之影響------------- 66
表14. HSP70基因多態型對產蛋性能之影響------------- 67



圖次
頁次
圖1. 緊迫程度對動物性能表現的影響----------------- 3
圖2. 緊迫啟動免疫系統、自主神經系統、神經內分泌系統
和行為反應--------------------------------- 4
圖3. 緊迫反應分為3個階段------------------------- 6
圖4. 熱緊迫造成行為、自主神經系統及內分泌系統的主要
反應影響生長與產蛋性能---------------------- 8
圖5. 雞隻於環境溫度43℃,喘氣狀態之體內溫度------- 11
圖6. 甲狀腺功能--------------------------------- 13
圖7. T3與T4參與光照週期之訊息傳遞影響雞隻繁殖性能-- 15
圖8. 輕微增加溫度造成果蠅唾液腺染色體膨大--------- 17
圖9. 細胞合成熱休克蛋白質的因子------------------- 18
圖10. HSP70蛋白質結構--------------------------- 21
圖11. HSP70反應的循環--------------------------- 22
圖12. HSP70主要的功能--------------------------- 25
圖13. 各階段工作-------------------------------- 28
圖14. 基因序列第258位點發生變異------------------ 37
圖15. 雞群HSP70基因型鑑定結果-------------------- 38
圖16. 熱處理溫度及時間對雞隻呼吸頻率之影響-------- 41
圖17. 熱處理溫度及時間對雞隻血漿CK活性之影響------ 42
圖18. 熱處理溫度與時間對雞隻T3濃度之影響---------- 43
圖19. HSP70基因型對熱緊迫雞隻呼吸頻率之影響------- 50
圖20. HSP70基因型對熱緊迫雞隻血漿CK活性之影響----- 55
圖21. HSP70基因型對熱緊迫雞隻血漿T3濃度之影響----- 58
圖22. 熱處理42℃達2小時及45℃達1小時之雞隻腿肉HSP70
mRNA表現量-------------------------------- 61
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