臺灣博碩士論文加值系統

熱緊迫使畜禽生長與生殖性能低落，嚴重熱緊迫會增加動物死亡率。下視丘為禽類體溫調節中樞，可以偵測外在環境溫度變化。表觀遺傳學為不改變DNA序列前提下透過鹼基上的化學修飾影響基因表現，啟動子上的DNA甲基化修飾通常被認為和基因靜默有關，環境熱緊迫也可能改變動物體內表觀遺傳修飾而使動物耐過或適應不利生存的環境。目前尚缺乏急性熱緊迫對於禽類下視丘基因表現與表觀遺傳影響之研究，本研究之目的為探討急性熱緊迫對L2品系台灣土雞公雞下視丘基因表現之影響以及DNA甲基化修飾是否造成基因差異表現。本研究使用197隻30週齡L2品系台灣土雞公雞，於熱緊迫實驗前逢機挑選五隻作為對照組，飼養在25℃且相對溼度55%環控氣候室；其餘192隻公雞接受38℃、相對溼度55%的急性熱緊迫四小時；熱緊迫處理後，所有公雞會依據體溫差(加熱期間體溫最大值減去加熱前體溫)分成熱敏感組(體溫差≧6.5℃)與熱抵抗組(體溫差≦2.5℃)，在試驗結束後每組取五隻雞採其下視丘樣品供後續分析。L2品系公雞下視丘急性熱緊迫後之mRNA表現、DNA甲基化程度定量與定性分別以RNA定序(RNA sequencing, RNA-seq)、焦磷酸定序(pyrosequencing, Pyro-seq)與甲基化特異性PCR (methylation specific PCR, MS-PCR)進行。急性熱緊迫處理後公雞下視丘差異表現基因(differentially expressed genes, DEGs)中有1,690個基因表現量顯著增加與819個基因表現量顯著減少；經生物功能(gene ontology)註解後可知此等差異表現基因主要參與細胞過程、代謝過程及生物性調節；急性熱緊迫顯著改變下視丘中熱緊迫蛋白家族(HSPB1、HSPB8、HSPB9及DNAJA4)、抗氧化酶(GPX1)、下視丘緊迫調節(FKBP5)與DNA甲基化與轉錄因子結合蛋白(DNMT3A及CREBBP)之表現量。MS-PCR結果顯示HSPB1在三組雞隻均為部分甲基化，HSPB8則皆為高度甲基化；HSPB9在對照組與熱敏感組雞隻為部分甲基化，在熱抵抗組雞隻為低度至部份甲基化；GPX1在對照組與熱敏感組雞隻為部分甲基化，在熱抵抗組雞隻為低度至部分甲基化。Pyro-seq結果顯示熱敏感組雞隻HSPB8、GPX1與DNMT3A啟動子內特定CpG位點之甲基化程度顯著高於抵抗組者。由本研究結果可知，急性熱緊迫可能改變台灣土雞公雞下視丘與緊迫調節、細胞凋亡、下視丘功能及抗氧化酶相關基因mRNA表現而保護下視丘免於急性熱緊迫帶來的損傷；Pyro-seq結果與RNA-seq部分呈現矛盾的現象，其原因可能為本研究分析之序列並非轉錄因子結合之位點，所以該位點甲基化程度的增加並不一定會降低該基因之表現，因此，該序列是否為轉錄因子結合位點有待進一步探討。

Heat stress causes poor growth performance, reproductive performance, and high mortality in farm animals. The hypothalamus acts as a crucial center to regulate body temperature and detect environmental temperature changes. Epigenetics is the study of genomic structural modifications that affects gene expression without altering the underlying nucleotide sequence. DNA methylation modifications on the CpG sites of promoter are generally thought to be related to gene silencing, and heat stress may alter epigenetic modifications to allow animals withstand or adapt to the environment stress. The purpose of this study was to investigate the effect of acute heat stress on gene expression and to explore whether the modification of DNA methylation affects gene expression in layer-type L2 strain Taiwan country chickens (TCCs). A total of 197 30-week-old roosters of L2 strain TCCs were used in this study. One hundred and nighty-two roosters were acute heat-stressed at 38°C and 55% RH for 4 h. After acute heat stress, the roosters were grouped according to the body temperature change (highest body temperature during heat stress minus pre-heat stress body temperature, ΔT), including sensitive group (Sen.) with ΔT≧6.5 and resistant group (Res.) with ΔT≦2.5. The other five roosters were kept at 25°C and 55% RH as a control group (Ctl.). The hypothalamus of five roosters in each group were collected for RNA sequencing (RNA-seq) and DNA methylation analysis by pyrosequencing (Pyro-seq) and methylation specific PCR (MS-PCR). There were 1,690 genes upregulated and 819 genes downregulated among the differentially expressed genes (DEGs) after acute heat stress in the hypothalamus of L2 strain TCCs. Gene ontology annotation indicated that most of the DEGs were involved in biological processes of cellular process, metabolic process and biological regulation. The expressions of genes in HSP family (HSPB1, HSPB8, HSPB9 and DNAJA4), antioxidant (GPX1), hypothalamus function (FKBP5), and DNA methylation and transcription (DNMT3A and CREBBP) were significantly changed and were selected for further DNA methylation analysis. Result of MS-PCR revealed that HSPB1 was partially methylated and HSPB8 was highly methylated in the three groups of roosters; HSPB9 was partially methylated in CTL group and SEN group and hypo- to partially methylated in RES group; GPX1 was partially methylated in CTL group and SEN group and hypomethylated to partially methylated in RES group. Pyro-seq revealed that the methylation level of specific CpG site in HSPB8、GPX1 and DNMT3A were higher in SEN group than RES group. In summary, acute heat stress changes the expression of genes related to the response to stimuli, antioxidant, and hypothalamus function of hypothalamus in TCCs. Moreover, some of the Pyro-seq results were partially conflict with the RNA-seq results. The possible reason may be that the sequences analyzed are not the transcription factor binding sites, thus the increase of methylation level did not reduce the level of gene expression. Therefore, whether the sequences are transcription factor binding sites require further exploration.

中文摘要.................................................i
Abstract...............................................ii
表次...................................................vi
圖次..................................................vii
壹、前言................................................1
貳、文獻探討............................................2
一、緊迫定義與熱緊迫.....................................2
二、雞隻體溫調節.........................................2
三、熱緊迫對雞隻生理與代謝的影響...........................3
四、下視丘與體溫調節.....................................3
(一)下視丘的位置與結構...................................3
(二)下視丘與體溫調節.....................................4
五、雞隻熱緊迫反應之功能性基因體學探討.....................5
六、表觀遺傳學定義與禽類DNA甲基化之研究現況................7
(一)表觀遺傳學(Epigenetics).............................8
(二)DNA甲基化(DNA methylation)..........................8
(三)DNA甲基化在禽類的研究現況............................10
七、研究目的...........................................11
參、材料方法...........................................12
一、試驗動物飼養管理....................................12
二、急性熱緊迫試驗與雞隻分群.............................12
三、雞隻下視丘RNA樣品製備與分析..........................13
(一)下視丘RNA的萃取...................................13
(二)RNA定序(RNA-Sequencing, RNA-seq)分析..............13
(三)差異表現基因(Differentially expressed genes, DEG)的挑選.....................................................13
(四)差異表現基因之生物功能分析...........................13
(五)即時定量聚合酶連鎖反應(Quantitative real-time polymerase chain reaction, qRT-PCR)...............................14
四、雞隻下視丘DNA樣品製備與分析...........................14
(一)下視丘DNA的萃取.....................................14
(二)DNA濃度與品質檢測...................................15
(三)DNA經重亞硫酸鹽轉換(DNA bisulfite conversion)........15
(四)甲基特異性聚合酶鏈鎖反應分析(Methylation specific PCR, MS-PCR)................................................17
(五)焦磷酸定序分析(Pyrosequencing, pyro-seq).............17
五、統計分析............................................20
肆、結果................................................24
一、急性熱緊迫後各組雞隻體溫差結果之比較...................24
二、急性熱緊迫對L2品系台灣土雞公雞下視丘基因表現之影響 .......................................................25
三、急性熱緊迫後L2品系台灣土雞公雞下視丘表現量增加基因之生物功能註解...................................................26
四、急性熱緊迫後L2品系台灣土雞公雞下視丘表現量減少基因之生物功能註解...................................................26
五、以qRT-PCR驗證基因之差異表現..........................26
六、挑選差異表現兩倍以上的DEGs經MS-PCR分析之結果...........31
七、差異表現基因焦磷酸定序(pyrosequencing)分析之結果.....................................................33
伍、討論................................................40
一、急性熱緊迫對L2品系台灣土雞公雞下視丘基因表現之影響......40
(一)急性熱緊迫影響熱緊迫蛋白質(heat shock proteins, HSPs)相關基因.................................................40
(二)急性熱緊迫影響下視丘中抗氧化相關基因...................41
(三)急性熱緊迫影響下視丘與緊迫反應相關基因之表現............42
(四)急性熱緊迫影響轉錄相關基因之表現.......................42
(五)急性熱緊迫影響細胞凋亡相關途徑........................43
二、受急性熱緊迫影響基因之DNA甲基化修飾....................45
(一)RNA-seq結果與pyrosequencing結果比較..................45
(二)MS-PCR結果與pyrosequencing結果比較...................46
陸、結論................................................49
柒、參考文獻............................................50
捌、附表................................................59

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