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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張俊哲(Chun-Che Chang) | |
dc.contributor.author | Yi-Tsen Lin | en |
dc.contributor.author | 林羿岑 | zh_TW |
dc.date.accessioned | 2021-06-16T09:32:30Z | - |
dc.date.available | 2022-02-17 | |
dc.date.copyright | 2017-02-17 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-02-14 | |
dc.identifier.citation | Agriculture and Food Agency (2013). Agricultural Statistics Yearbook. Agriculture and Food Agency, Council of Agriculture, Executive Yuan, Taiwan.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59671 | - |
dc.description.abstract | 生殖細胞為專司遺傳訊息傳遞至子代之細胞。已知部分動物如黑腹果蠅,生殖細胞特化通常發生於發育的早期,其形成需倚賴聚集在卵室後端的細胞質,又稱生殖漿。母系遺傳的生殖細胞決定因子表現於生殖漿,並於胚胎發育時期促使後端形成生殖細胞 (極細胞)。在眾多生殖細胞決定因子中,oskar 基因扮演招攬並固定其他生殖漿組成物於後端之上游角色。oskar 基因產物表現於卵發育與早期胚胎發育的後端,此現象已被證實保守於部分完全變態昆蟲中,如蚊和蜂,因而推論 oskar 基因產物可標定卵發育時期的生殖漿形成。根據推論,我們選殖和黑腹果蠅同屬雙翅目之東方果實蠅 oskar 同源基因 (Bdosk),並偵測卵發育時期的基因表現。透過整體原位雜合法我們發現 Bdosk mRNA 的表現模式與黑腹果蠅 Dmosk mRNA 相似,皆表現於卵細胞的後端。而螢光免疫染色結果呈現 BdOsk 蛋白表現模式同樣與 DmOsk 蛋白相似,證實 oskar 基因於雙翅目昆蟲中保守參與生殖漿組裝。為了驗證 oskar 基因作為分子錨的保守性,我們透過 UAS-Bdosk-Dmbcd 3’ UTR 轉基因黑腹果蠅,異位表現 Bdosk mRNA 與蛋白質於黑腹果蠅卵前端。我們成功偵測到 BdOsk 的異位表現,然還未進一步觀察是否可誘導異位生殖細胞於黑腹果蠅胚胎前端產生。儘管如此,我們的研究為 Bdosk 於東方果實蠅卵發育表現的研究奠定基礎,並貢獻於雙翅目昆蟲 oskar 基因演化角色之探討。 | zh_TW |
dc.description.abstract | Germ cells, by definition, are the cells that can transmit genetic information from generation to generation. In some animals such as the fruit fly Drosophila melanogaster, germ cells are specified at the beginning of development, relying on a specialized cytoplasm called germ plasm. Maternally-inherited germline determinants restricted within the germ plasm are then recruited to the germ (pole) cells that are formed in the posteriormost region of the embryo after fertilization. Among all maternal germline determinants in Drosophila, oskar (osk) mRNA is known to act as molecular anchors to recruit other components of the germ plasm. Posterior localization of osk mRNA in oocytes and early eggs have also been identified in several other holometabolous insects such as mosquitos and wasps, suggesting that restriction of osk transcripts in the developing oocytes labels the forming germ plasm. Based upon this assumption, I cloned an osk ortholog (Bdosk) in oriental fruit fly Bactrocera dorsalis, detecting its expression during oogenesis. Data of in situ hybridization showed that posterior localization of Bdosk mRNA, like that of Drosophila osk, was identified in the posterior poles of the developing oocytes. Localization pattern of the BdOsk protein resembled to that of Bdosk mRNA, indicating that osk plays a conserved role to assemble the germ plasm in the Diptera. In order to verify to which extent the role as a molecular anchor is conserved, we ectopically expressed Bdosk mRNA and protein to the anterior pole of Drosophila oocytes with the aid of the 3’UTR of bicoid (bcd), a noncoding region known to direct bcd mRNA to the oocyte anterior. Anterior localization of BdOsk has been identified but we have not been able to observe whether Bdosk can induce the formation of pole cells in the anterior region of the Drosophila embryos. Notwithstanding, our study paves a path for further investigation of the evolutionary role of osk in the Diptera. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T09:32:30Z (GMT). No. of bitstreams: 1 ntu-106-R03b48002-1.pdf: 113387842 bytes, checksum: cd571b3d06a6dc58039804715840179c (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 口試委員會審定書 i
目錄 ii 致謝 vi 表目錄 vii 圖目錄 viii 中英對照表 x 中文摘要 xii Abstract xiii 第一章、前言 1 1. 生殖細胞 1 1.1 生殖細胞發育概述 1 1.2 黑腹果蠅生殖漿組裝與生殖細胞特化 2 2. 黑腹果蠅卵發育 3 3. oskar 基因 4 3.1 oskar 基因序列 4 3.1.1 Long Oskar 與 Short Oskar 4 3.1.2 LOTUS 結構域 5 3.1.3 SGNH hydrolase 結構域 / OSK 結構域 6 3.2 oskar 表現模式 6 3.3.1 oskar mRNA 表現模式與調控 6 3.3.2 Oskar 蛋白表現模式與轉譯調控 7 3.3 Oskar在其他昆蟲的往昔研究探討其演化上所扮演的角色 7 3.3.1 oskar 基因於生殖漿組裝所扮演之角色 8 3.3.2 oskar 基因於神經發育所扮演之角色 9 4. 東方果實蠅生物學 10 4.1 東方果實蠅生活史 10 4.2 東方果實蠅的危害 10 4.3 東方果實蠅於雙翅目之演化地位 11 5. 東方果實蠅 RNA 干擾技術 11 6. 研究動機與目的 12 第二章、材料與方法 14 1. 東方果實蠅的飼養 14 2. 東方果實蠅卵巢取得與解剖 14 3. 體原位雜合法 15 3.1 探針製備 15 3.1.1 萃取全 RNA 和反轉錄 15 3.1.2 設計專一性引子 16 3.1.3 聚合酶連鎖反應與產物回收 16 3.1.4 接合反應與質體轉形至 E. coli JM109 品系 17 3.1.5 養菌 18 3.1.6 質體萃取 18 3.1.7 限制酶截切使質體線性化 20 3.1.8 DNA 沉降 20 3.1.9 胞外轉錄 20 3.1.10 RNA 沉降 20 3.2 整體原位雜合法 21 3.2.1 東方果實蠅 / 黑腹果蠅卵巢固定、脫水與復水 21 3.2.2 卵巢與蛋白酶 K 作用增加探針穿透性 22 3.2.3 卵巢再次固定 22 3.2.4 探針雜合反應 22 3.2.5 酵素受質呈色 23 3.2.6 辨識細胞核之螢光抗體染色 23 4. 蛋白質表現 23 4.1 引子設計和接合反應 23 4.2 C41 勝任細胞處理和轉形 24 4.3 蛋白質小量表現 25 4.4 蛋白質大量表現 26 4.5 蛋白質純化 27 4.5.1 可溶性測試 27 4.5.2 親和層析法 28 4.5.3 SDS-PAGE 膠體蛋白質電泳 30 5. 胜肽多株抗體製備 31 6. 西方墨點法 32 6.1 東方果實蠅卵巢蛋白質萃取 32 6.2 西方墨點法 32 7. 螢光免疫染色 33 8. DAPI 和 Phalloidin 染色 34 9. 牛小腸磷酸酶作用 34 10. RNA 干擾技術 35 10.1 dsRNA 製備 35 10.2 顯微注射針製備 36 10.3 東方果實蠅顯微注射 36 10.4 反轉錄聚合酶鏈鎖反應 37 第三章、實驗結果 38 1. 基因選殖 38 1.1 東方果實蠅 oskar 基因選殖與探針製備 38 1.2 東方果實蠅 α-Tubulin 基因選殖 38 2. 原位雜合法觀察 Bdosk mRNA 於東方果實蠅微卵管中的表現 39 3. 原位雜合法比較 Bdosk 與 Dmosk mRNA 於微卵管的表現差異 39 4. 東方果實蠅 BdOsk_SGNH domain 表現載體建構及融合蛋白表現 40 5. 東方果實蠅 BdOsk 蛋白質純化 42 6. 東方果實蠅 BdOsk 胜肽合成與多株抗體製備 44 7. 東方果實蠅 Oskar 西方墨點法 45 7.1 BdOsk_non-conserved domain 表現載體建構及融合蛋白表現 45 7.2 BdOsk_non-conserved domain 蛋白質純化 46 7.3 西方墨點法偵測東方果實蠅 BdOsk 抗體專一性 47 8. 螢光免疫染色分析東方果實蠅微卵管 Oskar 表現與分佈 48 9. 比較不同發育時期卵細胞 BdOsk 與 DmOsk 蛋白表現形式之異同 48 10. 根據核染色鑑別東方果實蠅卵發育時期 50 11. UAS-Bdosk-Dmbcd 3’ UTR 轉基因黑腹果蠅卵前端表現 BdOsk 51 12. Bdosk dsRNA 與 EGFP dsRNA 製備 53 13. 東方果實蠅腹部顯微注射 54 14. 透過 RNA 干擾抑制東方果實蠅 Bdosk 基因表現 55 第四章、討論 57 1. 東方果實蠅卵發育時期定義並與黑腹果蠅卵發育時期比較 57 2. Bdosk mRNA 於東方果實蠅微卵管中的表現模式 60 3. BdOsk 抗體製備與西方墨點法 61 4. 東方果實蠅卵發育過程中 Bdosk 蛋白轉譯早於黑腹果蠅 62 4.1 Bdosk mRNA 提早轉譯但未表現於後端的 mRNA 仍被轉譯抑制 63 4.2 Bdosk mRNA 提早轉譯可能與去轉譯抑制機制的提早啟動有關 63 4.3 東方果實蠅的轉譯活化可能受到空間信號的驅動 64 4.4 東方果實蠅 BdOsk 的卵前端表現探討 65 5. RNA 干擾抑制東方果實蠅 Bdosk 基因表現實驗檢討與改進 65 6. UAS-Bdosk-Dmbcd 3’ UTR 轉基因黑腹果蠅 Bdosk mRNA 表現模式 66 7. 東方果實蠅 oskar 同源基因表現研究的貢獻與延展 67 附錄一、引子對序列資訊 134 附錄二、藥品配置 137 附錄三、口試問答紀錄 141 參考文獻 145 | |
dc.language.iso | zh-TW | |
dc.title | 解析東方果實蠅 oskar 基因產物在卵後端之聚集與生殖漿的形成 | zh_TW |
dc.title | Posterior localization of oskar mRNA and protein evidences the assembly of germ plasm during oogenesis in the oriental fruit fly Bactrocera dorsalis | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林明德(Ming-Der Lin),蕭信宏(Shin-Hong Shiao),江運金(Yun-Jin Jiang) | |
dc.subject.keyword | 卵發育,微卵管,生殖細胞決定物質,雙翅目,胚胎發育, | zh_TW |
dc.subject.keyword | oogenesis,ovarioles,germline determinants,dipteran,embryogenesis, | en |
dc.relation.page | 152 | |
dc.identifier.doi | 10.6342/NTU201700608 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-02-15 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 基因體與系統生物學學位學程 | zh_TW |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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