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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張俊哲 | |
| dc.contributor.author | Yao-chung Chen | en |
| dc.contributor.author | 陳躍中 | zh_TW |
| dc.date.accessioned | 2021-06-08T02:12:17Z | - |
| dc.date.copyright | 2016-02-24 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2016-01-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19666 | - |
| dc.description.abstract | 豌豆蚜 (Acyrthosiphon pisum) 受日照時間的改變刺激會有有性生殖和無性生殖的模式。有性及無性生殖模式成蟲的微卵管型態具有獨特發育多型性，無性胎生微卵管內含有卵及胚胎；而有性卵生微卵管只有充滿卵黃的未受精卵。Gallot et al (2012) 檢測 33 個基因在有性及無性微卵管的卵細胞中有不同的表現，推論這些基因有一些同時參與有性及無性卵發育的分化，有一些只有參與有性或是無性卵發育。然而 33 個基因當中有 7 個卵生成基因被表現在無性胎生豌豆蚜胚胎發育 Stage 6 的初始生殖細胞中，分別是：ApGle1、ApBic-C、ACYPI25088 (Trf4-like)、ACYPI010052、ACYPI007465、ACYPI39770 以及 ACYPI54656。這些卵生成基因是否參與生殖細胞的發育還未知。ApGle1、ApBic-C 以及 ACYPI25088 (Trf4-like) 推測參與 mRNA polyadenylation，而其他 4 個卵生成基因為蚜蟲特有的基因。我採用整體原位雜合技術檢測 7 個卵生成相關的基因在胚胎發育時期的表現，實驗結果為無性豌豆蚜生殖細胞在胚胎發育過程中，這些卵生成基因全都被專一地表現在早期胚胎中。其中 5 個 ApBic-C、ACYPI25088 (Trf4-like)、ACYPI010052、ACYPI007465、ACYPI39770 被表現在移動中的生殖細胞以及後期胚胎的生殖細胞，而 ApGle1 亦被表現在後期胚胎的生殖細胞中。根據實驗結果推測無性豌豆蚜卵生成基因可能與胚胎時期的生殖細胞發育有關。 | zh_TW |
| dc.description.abstract | Pea aphid (Acyrthosiphon pisum) display parthenogenetic viviparous and sexual oviparous phases of reproduction in response to the change of photoperiods. Morphological structures of the ovarioles in asexual and sexual aphids display distinct developmental polyphenism – egg chambers of the parthenogenetic viviparous ovarioles accommodate oocytes and embryos whilst the sexual oviparous ovarioles only contain unfertilized eggs full of yolk. Gallot et al (2012) identified 33 genes differentially expressed in developing oocytes in asexual and sexual pea aphids, suggesting that some of these genes are involved in the differentiation of asexual and sexual oogenesis. However, there are 7 oogenesis genes from 33 genes are expressed in the primordial germ cells at stage 6 in asexual embryos including: ApGle1、ApBic-C、ACYPI25088 (Trf4-like)、ACYPI010052、ACYPI007465、ACYPI39770 and ACYPI54656. Whether they are involved in germline development remain unknown. Analyze the protein sequence of ApGle1、ApBic-C、ACYPI25088 (Trf4-like) suggested they might be involved in mRNA polyadenylation and the remain 4 genes are suggested to be aphid-specific genes. I detected expression of 7 oogenesis genes in embryos and analyzed whether they were specifically restricted to germ cells by whole-mount in situ hybridization. Results show all of them are expressed in early embryos. 5 of them, ApBic-C、ACYPI25088 (Trf4-like)、ACYPI010052、ACYPI007465 and ACYPI39770 are expressed in the migrating germ cells and germ cells in somatic gonads. ApGle1 is expressed in germ cells during late embryogenesis. Results show oogenesis gene may play a role in embryonic germline development in asexual pea aphids. | en |
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| dc.description.tableofcontents | 目錄
摘要 i Abstract ii 第一章 前言 1 1.1豌豆蚜的生物特性 1 1.2 豌豆蚜的卵生成 2 1.3 無性豌豆蚜的生殖細胞發育 4 1.4 Gle1 基因的簡介 5 1.5 Bicaudal-C 基因的簡介 6 1.6 研究動機 7 1.7 研究目的 7 第二章 材料與方法 8 2.1 豌豆蚜蟲的飼養 8 2.2 合成探針 8 2.2.1 萃取 total RNA 8 2.2.2 RNA 反轉錄成 cDNA 9 2.2.3 設計專一性引子 9 2.2.4 PCR 操作和產物回收 10 2.2.5 PCR 產物電泳分析 11 2.2.6 PCR 產物回收 11 2.2.7 接合到載體及轉型到勝任細胞 11 2.2.8 養菌及質體萃取 12 2.2.9 限制酶反應 DNA 沉澱 14 2.2.10 DNA 沉澱反應 14 2.2.11 胞外轉錄 (in vitro transcription) 14 2.2.12 RNA 沉降 15 2.3 原位雜合反應 (Whole mount in situ hybridization) 15 2.3.1 固定 (Fixation) 15 2.3.2 再水合 (Rehydration) 16 2.3.3 雜合前處理 (Prehybridization) 16 2.3.4 雜合反應 (Hybridization) 16 2.3.5 清洗 (Wash) (清洗溫度為 65℃) 16 2.3.6 偵測探針 (Detection) 17 2.3.7 偵測 DIG 標記探針 (Detection of DIG-labelled probe) 17 2.4 微卵管 RNA 抽取與反轉錄 PCR 17 2.4.1 使用 TRIzol 抽取微卵管 RNA 17 2.4.2 使用反轉錄 PCR 檢測微卵管中 ACYPI54656 RNA 的表現量 18 第三章 實驗結果 19 3.1 基因的選殖 19 3.1.1 ApGle1 的選殖 19 3.1.2 ApBic-C 的選殖 19 3.1.3 Hypothetical gene 的選殖 20 3.3 整體原位雜合法結果 24 3.3.1 ApGle1 mRNA 在微卵管的表現分析 24 3.3.2 ApBic-C mRNA 在微卵管的表現分析 25 3.3.3 ACYPI25088 mRNA在微卵管的表現分析 26 3.3.4 ACYPI010052 mRNA 在微卵管的表現分析 27 3.3.5 ACYPI39770 mRNA 在微卵管的表現分析 28 3.3.6 ACYPI007465 mRNA在微卵管的表現分析 29 3.3.7 ACYPI54656 mRNA在微卵管的表現分析 30 第四章 討論 33 4.1 實驗結果與 Gallot et al (2012) 文獻的比較 33 4.1.1 基因的選殖結果與 Gallot et al (2012) 文獻的比較 33 4.1.2 整體原位雜合反應與 Gallot et al (2012) 文獻的比較 34 4.2 卵生成基因與 Polyadenylation 的關係 35 4.3 豌豆蚜卵生成基因在胚胎發育過程中的表現 37 4.3.1 ApGle1 在胚胎發育之表現分析 37 4.3.2 ApBic-C在胚胎發育之表現分析 38 4.3.3 ACYPI25088 (Trf4-like) 在胚胎發育之表現分析 39 4.3.4 ACYPI010052在胚胎發育之表現分析 40 4.3.5 ACYPI39770、ACYPI007465 以及 ACYPI54656在胚胎發育之表現分析 41 4.4 卵生成基因的表現與移動中的生殖細胞的關係 43 4.5 重要性與未來展望 44 參考文獻 46 圖一、豌豆蚜生活史 53 圖二、無性胎生豌豆蚜胚胎發育時期的定義 54 圖三、7 個被表現在無性豌豆蚜第 6 時期生殖細胞的基因 57 圖四、無性豌豆蚜的生殖細胞發育 58 圖五、ApGle1 的選殖 61 圖六、ApBic-C 選殖區域與預測片段的核苷酸及胺基酸序列的比較 63 圖七、ApBic-C 轉型到 pGEM-Teasy vector 之示意圖 65 圖八、ACYPI25088 的選殖序列比較 66 圖九、NT_PAP_TUTase domain 以及 PAP_assoc domain 之胺基酸序列在各物種 之間的比較 68 圖十、ACYPI25088 轉型到 pGEM-Teasy vector 之示意圖 69 圖十一、ACYPI010052 選殖區域與預測片段胺基酸序列的比較 70 圖十二、ACYPI010052 轉型到 pGEM-Teasy vector 之示意圖 72 圖十三、ACYPI39770 的選殖 74 圖十四、ACYPI007465的選殖 77 圖十五、ACYPI54656的選殖 79 圖十六、偵測 ApGle1 mRNA 在豌豆蚜卵細胞和胚胎發育的表現 80 圖十七、偵測 ApBic-C mRNA 在豌豆蚜卵細胞和胚胎發育的表現 82 圖十八、偵測 ACYPI25088 mRNA在豌豆蚜卵細胞和胚胎發育的表現 84 圖十九、偵測 ACYPI010052 mRNA在豌豆蚜卵細胞和胚胎發育的表現 86 圖二十、偵測 ACYPI010052 mRNA 在豌豆蚜胚胎翻轉 (Katatrepsis) 的表現 88 圖二十一、偵測 ACYPI39770 mRNA 在豌豆蚜卵細胞和胚胎發育的表現 89 圖二十二、偵測 ACYPI007465 mRNA 在豌豆蚜卵細胞和胚胎發育的表現 91 圖二十三、偵測 ACYPI54656 mRNA 在豌豆蚜卵細胞和胚胎發育的表現 93 圖二十四、偵測 ACYPI54656 mRNA 在豌豆蚜一齡若蟲微卵管的表現 95 圖二十五、使用反轉錄 PCR 檢測 ACYPI54656 mRNA 在微卵管的表現分析 96 表一、33 個在有性以及無性胚胎表現量有差異的基因描述 (Gallot et al., 2012) 97 表二、7 個卵生成基因的專一性引子序列 98 表三、ACYPI25088 比對資料庫整理表 99 表四、ACYPI010052 比對資料庫整理表 105 表五、ACYPI39770 比對資料庫整理表 114 表六、ACYPI007465 比對資料庫整理表 116 表七、ACYPI54656比對資料庫整理表 121 表八、基因選殖結果以及蛋白保守區域之預測功能與 Gallot et al (2012) 文獻的 比較整理 125 表九、基因表現形式與 Gallot et al (2012) 文獻的比較整理 127 | |
| dc.language.iso | zh-TW | |
| dc.title | 豌豆蚜卵生成基因在胚胎發育之表現分析 | zh_TW |
| dc.title | Analysis of the expression of oogenesis genes during embryogenesis in the pea aphid Acyrthosiphon pisum | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蕭信宏,劉逸軒,林明德 | |
| dc.subject.keyword | 豌豆蚜,卵生成基因,生殖細胞發育,Gle1,Bicaudal-C,Trf4,mRNA polyadenylation, | zh_TW |
| dc.subject.keyword | Pea aphid,Oogenesis genes,Germline development,Gle1,Bicaudal-C,Trf4,mRNA polyadenylation, | en |
| dc.relation.page | 137 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2016-01-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 昆蟲學研究所 | zh_TW |
| 顯示於系所單位： | 昆蟲學系 | |
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