臺灣博碩士論文加值系統

每年雞蛋從生產到運輸至消費者手中，約有13-20 % 的破損率，若增加其蛋殼強度將可減少其經濟損失。褐色菜鴨是本省產蛋率高且蛋殼品質優於雞蛋的家禽，其蛋殼強度呈現中度遺傳（0.53），在高蛋殼強度品系的蛋殼強度顯著比低蛋殼強度品系優異。近年來有關蛋殼品質的分子層次之研究，皆為蛋殼之有機基質，雖有機基質只占整個蛋殼的1~2%，卻是蛋殼品質的關鍵點。
本研究旨在針對褐色菜鴨在產蛋後之特定時段，比較蛋殼強度不同鴨隻之輸卵管殼腺部黏膜層細胞的基因表現情形，並嘗試選殖及定序彼等可能涉及蛋殼品質之基因，冀對於蛋殼品質之分子影響機制，能有更深入之瞭解。試驗初始乃依蛋殼強度之測定記錄，將褐色菜鴨分為強蛋殼（HES；ES>5.5 kg）與弱蛋殼（LES；ES>3.5 kg）兩大類，其二者經過分析後，的確是有強度上之差異（p< 0.0001）。鑑於褐色菜鴨排出之卵進入殼腺部的時刻，已知係在前一枚蛋產下後約5~7 h；推斷在此一時段前夕，當係蛋殼乳突層形成且係蛋殼有機基質開始迅速合成與堆積之際，進一步試驗及針對HES與LES鴨隻，分別於產蛋後4.5小時階段，先經過檢查確認其生殖道內存在有一枚形成中之蛋者，即將之犧牲並儘速刮取其管狀殼腺部之黏膜層細胞，經分別抽取其總RNA後，並利用72組引子組合，分別進行mRNA之差異展現分析（differential display polymerase chain reaction；DDRT-PCR）與two-step DDRT-PCR分析，俾比較HES與LES管狀殼腺部（tubular shell gland；TSG）之黏膜層細胞在這時段對於mRNA基因表現之差異性。試驗結果合計獲得16個具蛋殼強度特異性之基因片段；其中11條和5條差異性片段分別來自DDRT-PCR與two-step DDRT-PCR。並將所有差異性片段，經完成選殖與核酸定序並於NCBI網站所提供之基因庫進行序列比對後，証明其中13條係新發現的基因。並挑選其中1條基因片段TG36S，進一步應用同步定量聚合連鎖反應（real-time quantitative polymerase chain reaction；QPCR）分析；試驗結果顯示，在HES的TSG之黏膜層細胞對於TG36S基因之轉錄量，確實較LES高約1.83倍。此外，就HES分析，菜鴨於生蛋後4.5 h時，TG36S基因在殼腺（SG）、TSG、峽部（isthmus）及蛋白分泌部（magnum）等部位之轉錄量，分別為其在峽部者之7.57、4.05與2.60倍。鑒於該特定時段適為新排出之卵進入子宮之前夕，前述基因在該時段特別於殼腺有極高之表現量是否意味其與蛋殼之形成有密切關係，則尚待完成其全長cDNA之定序與定性後，始克針對其可能扮演之生理學功能，進行深入之探討。

Deterioration of eggshell quality associated with many factors causes economic loss due to breakage during the egg transportation and processing. While many approaches including aspects related to nutrition, feeding management, or housing environment etc. have so far been employed years for enhancing the shell strength of chicken eggs, there are approximate 12% of all commercial eggshells broken or cracked between oviposition and consumption. The organic matrix, though it occupies only 1 ~ 2% of the whole eggshell contents, can be one of the most important components involved in eggshell quality. Therefore, in this present study, attempts were made to identify and characterize gene(s) differentially expressed 4.5 h post oviposition in epithelia of tubular shell-glands of brown Tsaiya and it is hopefully that the results will provide a better understanding to the molecular mechanisms related to eggshell formation.
To achieve the said purpose, two herds of brown Tsaiya ducks showing significant difference in eggshell strength (ES), the high eggshell strength (HES) with an average ES value over 5.5 kg and the low eggshell strength (LES) with an average ES value less than 3.5 kg, were employed in the present study. Total RNA samples were extracted from epithelial cells of tubular shell gland 4.5 h post-ovipostion. Each RNA sample was subjected to analysis by the method of differential display reverse transcription polymerase chain reaction （DDRT-PCR） and two-step DDRT-PCR with a total of 72 primer sets designed. Comparisons were made to identify specific fragments of cDNA(s) between each sample amplified after the DDRT-PCR and two-step DDRT-PCR analysis. Among the 72 pairs of primer sets tested, only 8 primer-pairs were found to be effective in amplifying specific DNA fragments from the diversified products amplified. A total of 16 differential fragments of cDNA were obtained, including 11 fragments from the method of DDRT-PCR and 5 fragments from the two-step DDRT-PCR method. One of them, named TG36S, was further subjected to quantitative comparisons of its mRNA expressed between herds of HES- and LES-ducks, with the method of Real-Time Quantitive polymerase chain reaction (QPCR). Results appeared that the amount of TG36S mRNA expressed in the tubular shell glands from HES-ducks was 1.83 fold higher than that in the same tissue from LES-ducks. Moreover, to those HES-ducks, it was also noticed that much higher expression of TG36S, with some 7.57, 4.05, and 2.60 folds higher based on the amount of TG36S mRNA expressed, was found in epithelia of shell glands, tubular shell glands, and magnum, respectively, when comparisons were made to that of in epithelia of isthmus. Based on the fact that much higher expression of TG36S found in HES-ducks, particularly in epithelia of shell glands and tubular shell glands, it is worth to further investigate the physiological function of this gene product.

目 錄...……………………………………………………………I
表 次……………………………………………………………...IV
圖 次……………………………………………………………...V
附錄圖次………………………………………………………...VII
摘 要…………………………………………………………... 1
緒 言……………………………………………………………...3
第 一 章 文 獻 討…………………………………………….5
1.1 研究目的及源起……………………………………………5
1.1.1 褐色菜鴨之簡介……………………………………….5
1.1.2 高蛋殼度與低蛋殼強度品系之比較………………….6
1.2 雌禽之生殖與生殖道的解剖構造及生理功能……………6
1.3 蛋的形成與結構分佈………………………………………13
1.3.1 蛋殼的形成過程……………………………………….13
1.3.2 蛋殼乳突層的顯微結構……………………………….13
1.4 蛋殼的成分與晶體形成之研究………………………………16
1.4.1 蛋殼的有機基質成分…………………………………. 16
1.4.2 蛋殼碳酸鈣晶體形成之探討………………………… 19
1.5 蛋殼強度之測定及影響因子……………………………….21
1.5.1蛋殼品質之測定方法………….……………………….21
1.5.2 影響蛋殼品質的因子………………………………….22
1.6 反轉錄聚合連鎖反應差異展現法(DDRT-PCR)………. 23
1.6.1 基本原理……………………………………………….23
1.6.2 影響因子……………………………………………….25
1.7 同步定量聚合連鎖反應（Real-Time quantitative polymerase chain reaction）……………………………………………25
1.7.1 基本原理……………………………...……………..26
1.7.2 △Rn、CT與threshold的定義………………………….27
1.7.3 引子及探針之設計條件………………………………..30
1.7.4 同步定量聚合連鎖反應溶液組成的特色…………..30
1.7.5 同步定量聚合連鎖反應的增幅條件…………...31
第 二 章 材 料 與 方 法…………………………………...32
2.1 樣品的收集……………………………………………….32
2.1.1 試驗鴨隻與蛋殼強度之測定…………………………..32
2.1.2 生殖道組織的收集…………………………………...32
2.2 褐色菜鴨管狀殼腺的黏膜上皮細胞中mRNA的差異展現分析34
2.2.1 total RNA的抽取……………………………………..34
2.2.2 確認total RNA濃度與品質………………………….35
2.2.2.1 迷你膠體電泳法…………………………………...35
2.2.2.2 RNA濃度的測定……………………………………35
2.2.3 利用DD-RT-PCR檢測特異性DNA片段……………….36
2.2.3.1 mRNA之反轉錄作用………………………………..36
2.2.3.2 聚合連鎖反應…………………………………….38
2.2.3.3 定序膠體電泳………………………………………38
2.2.3.4 回收特異性DNA表現之片段……………………..40
2.2.4 特異性片段的構築…………………………………...41
2.2.5 用PCR的方式來確認insert size………………………41
2.3 褐色菜鴨的部分β-actin基因序列的構築……………...43
2.3.1 引子的設計…………………………………………...43
2.3.2 PCR產物的築構…………………………………………43
2.3.3 基因序列的比對……………………………………...46
2.4 定量聚合連鎖反應（QPCR）……………………………46
2.4.1 資料分析……………………………………………...48
第 三 章 結 果 與 討 論…………………………………...50
3.1 試驗動物蛋殼強度之結果分析…………………………….50
3.2 錨定與隨機引子之配對組合對褐色菜鴨管狀殼腺黏膜上皮細胞基因轉錄物（mRNA）進行DDRT-PCR測試之效果.………………..52
3.2.1 於HES褐色菜鴨所選殖到的基因片段………………..54
3.2.2 於LES褐色菜鴨所選殖到的基因片段………………..71
3.3 褐色菜鴨管狀殼腺黏膜上皮細胞中表現具有蛋殼強度差異性基因之屬性……………………………………………………………...72
3.4 β-actin核酸序列的比對………………………………..75
3.5 QPCR的試驗結果…………………………………………..75
3.5.1 TG36S與β-actin的增幅效率………………………..77
3.5.2 目標基因（TG36S）在不同蛋殼強度的褐色菜鴨的比較77
第 四 章 結 論……………………………………………….87
第 五 章 參 考 文 獻………………………………………….88
英 文 摘 要……………………………………………………….96
附　　錄………………………………………………………...98
作 者 小 傳……………………………………………………….101

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