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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張俊哲(Chun-che Chang) | |
| dc.contributor.author | Gee-Way Lin | en |
| dc.contributor.author | 林季瑋 | zh_TW |
| dc.date.accessioned | 2021-06-08T06:27:57Z | - |
| dc.date.copyright | 2006-07-27 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-27 | |
| dc.identifier.citation | Balinsky, B. I. (1966). Changes in the ultrastructure of amphibian eggs following fertilization. Acta. Embryol. Morphol. Exp. 9, 132-154.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25747 | - |
| dc.description.abstract | 在大部分的動物中，胚胎發育早期生殖細胞即會與體細胞分離出來，以產生配子細胞。生殖細胞如何特化，它們如何移動至性腺，在模式昆蟲黃果蠅 (Drosophila melanogaster) 的研究最為詳盡。在果蠅中，初始生殖細胞 (極細胞) 的形成，依賴聚集於卵後端的生殖細胞決定物質，在多核單細胞之囊胚層形成前，包覆初始生殖細胞的細胞膜將生殖細胞決定物質納入細胞內，使得此類細胞具備發育成生殖細胞之潛力。不同於果蠅模式的非洲沙漠飛蝗 (Schistocerca gregaria) 的生殖細胞特化首度發現是於胚胎發育中期的腹部邊緣細胞。目前尚未發現蝗蟲的卵與早期胚胎有生殖細胞決定物質聚集的現象，因此其生殖細胞的特化較可能藉由鄰近體細胞之訊息分子誘發形成。選用孤雌生殖之豌豆蚜 (Acyrthosiphon pisum) 作為研究生殖細胞特化之材料，原因在於它早期形成囊胚層之模式與果蠅相似，然而其體節發育的方式卻類似於非洲沙漠飛蝗，因此研究此種昆蟲有助於明瞭不同發育形式的物種中，生殖細胞的特化是受到胚胎發育的那個階段所影響。研究策略的初步為先選殖豌豆蚜 vasa 同源基因，vasa 已被發現表現於多種無脊椎與脊椎動物的生殖細胞內，故推測它很有可能也是蚜蟲生殖細胞的標記基因，可作為追蹤生殖細胞的分子標記。本論文已完成兩種豌豆蚜 vasa 基因 (pavasa1 和 pavasa2) 之蛋白質表現與純化，並分別產生此兩種 Vasa 之抗血清。然而，不論是原位雜合或是免疫染色的結果都顯示 pavasa1 和 pavasa2的 mRNA 與蛋白質並非專一性地表現在已可辨識的生殖細胞當中。幸運地，還選殖到第 3 個豌豆蚜 vasa 同源基因 (pavasa3)，並且偵測到它的 mRNA 專一地表現在生殖細胞中，充分顯示pavasa3 為豌豆蚜之生殖細胞標記基因。 | zh_TW |
| dc.description.abstract | Germ cells are the cells that produce gametes, and they are segregated from somatic cells during early development in most animals. In the model insect Drosophila melanogaster, the specification of primordial germ cells (pole cells) depends on the maternal germline determinants localized to the egg posterior. By the formation of syncytial blastoderm, germline determinants are incorporated into pole cells with the forming cell membranes and then these cells are set to become germ cells. Unlike Drosophila, a maternal germ plasm is not identifiable in the grasshopper Schistocerca gregaria and segregated germ cells are first identified in the margin of abdomen undergoing segmentation, suggesting that germline specification may depend on induction signals released from neighboring somatic cells. I selected to study the parthenogenetic pea aphid Acyrthosiphon pisum because of its special pattern of embryogenesis - the formation of blastoderm resembles to Drosophila whilst the adding of segments in later development is similar to Schistocerca. It thus becomes an excellent model for investigating how germline specification is affected by different types of embryogenesis. Homologues of the Drosophila vasa gene are used as germline markers for this study because vasa is highly conserved in germ cells across many species across invertebrates and vertebrates. I expressed and purified fusion proteins of two pea aphid vasa homologues, pavasa1 and pavasa2, using them as antigens to induce anti-Vasa antiserum respectively. Nevertheless, immuno-staining results showed that no localized Vasa signals were identified in the germ cells. Likewise, in situ signals of localized pavasa1 and pavasa2 mRNAs were not germline positive. Fortunately I cloned pavasa3, the third vasa homologue in pea aphids, demonstrating that it was specifically expressed in germ cells. It strongly suggests that pavasa3 is a germline marker in pea aphids and it can be used as a molecular tool for studying germline development in this species. | en |
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| dc.description.tableofcontents | 中文摘要………………………………………………………………01
英文摘要………………………………………………………………02 前言……………………………………………………………………03 壹、生殖細胞之發育模式…………………………………………03 貳、昆蟲生殖細胞之發育…………………………………………04 參、利用 vasa 基因研究生殖細胞之發育模式…………………05 肆、研究豌豆蚜 vasa 基因以探討生殖細胞發育之策略………07 結果……………………………………………………………………09 壹、以跨物種交叉反應的 Formosa 抗體對豌豆蚜微卵管進行免疫染色…09 貳、Formosa 抗體與豌豆蚜全蛋白質進行西方墨點 (Western blot)…09 參、豌豆蚜 Pavasa1 與 Pavasa2 及非洲沙漠飛蝗 Sgv 蛋白質表現載體建構及融合蛋白表現…………………………………………09 肆、豌豆蚜 Pavasa1 與 Pavasa2 及非洲沙漠飛蝗 Sgv 蛋白質純化……10 伍、Formosa 抗體、豌豆蚜 Pavasa1、Pavasa2 及非洲沙漠飛蝗 Sgv 抗血清西方墨點法……………………………………………… 12 陸、豌豆蚜 Pavasa1、Pavasa2 及非洲沙漠飛蝗 Sgv 抗血清免疫染色……… 12 柒、豌豆蚜 pavasa3 基因選殖……………………………………13 捌、豌豆蚜體細胞與卵巢細胞 pavasa3 mRNA 表現量比較……14 玖、原位雜合以觀察 pavasa3 mRNA 於豌豆蚜微卵管內的表現………14 討論…………………………………………………………… 16 壹、豌豆蚜 3 個 Vasa-like 蛋白與其他昆蟲蛋白質序列比對分析………16 貳、豌豆蚜 3 個 vasa-like 基因與黃果蠅 vasa 基因 5’UTR 比對分析……… 17 參、Formosa 抗體所辨識的豌豆蚜蛋白可能為 Pavasa3 蛋白質………………18 肆、跨物種交叉性的 Formosa 抗體與豌豆蚜 Vasa 蛋白結合是依賴非洲沙漠飛蝗 Sgv 抗原特性……………………………… 18 伍、豌豆蚜 pavasa3 mRNA 與 Vasa 蛋白於微卵管中的表現…………… 19 陸、vasa 基因之表現與一物種具有多個 vasa 基因之探討……………19 柒、豌豆蚜 vasa 同源基因表現研究的貢獻與延展…………21 材料與方法…………………………………………………………23 壹、豌豆蚜的飼養………………………………………………23 貳、豌豆蚜卵巢 (ovary) 的取得……………………………23 參、豌豆蚜 pavasa1 與 pavasa2 基因之蛋白質表現載體建構…………24 一、Pavasa1 蛋白質表現載體…………………………24 二、Pavasa2 蛋白質表現載體…………………………25 肆、豌豆蚜 Pavasa1、Pavasa2 與非洲沙漠飛蝗 Sgv 融合蛋白之表現……25 一、表現菌落之建構與挑選……………………………25 二、蛋白質於菌體胞內表現……………………………26 三、蛋白質大量於菌體胞內表現之條件測試…………26 伍、豌豆蚜 Pavasa1、Pavasa2 與非洲沙漠飛蝗 Sgv 融合蛋白純化……27 一、測試融合蛋白的可溶性 (solubility)……… 27 二、製備表現融合蛋白之菌體細胞裂解液……………28 三、親合層析法-金屬螯合膠體的平衡及與細胞裂解液結合………28 四、親合層析法-金屬螯合膠體的流洗和融合蛋白溶出……… 28 五、以 SDS-PAGE 電泳分析純化之融合蛋白………… 29 六、濃縮純化之融合蛋白並經電泳切膠送以抗體製備…………29 陸、以豌豆蚜 Pavasa1、Pavasa2 與非洲沙漠飛蝗 Sgv 抗體進行免疫染色…… 30 一、樣本製備：解剖孤雌生殖豌豆蚜卵巢………………30 二、微卵管阻滯 (Blocking) 作用及與 Pavasa1、Pavasa2 與 Sgv 一次抗體結合………………………………………………30 三、微卵管與二次抗體作用………………………………31 四、酵素受質呈色…………………………………………31 柒、西方墨點法…………………………………………………32 一、樣本製備：卵巢內所有蛋白質與 Pavasa1、Pavasa2 蛋白抗原……32 二、蛋白質電泳轉印………………………………………32 三、轉印膜的阻滯及與跨物種交叉性反應的 Formosa 抗體作用………33 四、酵素受質呈色…………………………………………33 捌、豌豆蚜 pavasa3 基因選殖……………………………… 34 一、退化性引子 (degenerate primers) 設計…………34 二、以基因體 DNA 為模板進行選殖…………………… 34 三、以 RNA 反轉錄產生 cDNA 後為模板進行選殖………34 玖、豌豆蚜 pavasa3 基因 5’RACE (Rapid Amplification of DNA End)……35 一、萃取豌豆蚜 mRNA………………………………… 35 二、向內設計第二組 pavasa3 基因反向引子 (nested primer)………36 三、合成豌豆蚜pavasa3 cDNA……………………………36 四、pavasa3 cDNA 尾端黏合 polyA………………… 36 五、以 3’ 端帶有 polyA 的 pavasa3 cDNA 為模板向 5’ 端增幅………36 六、以 5’ 端延展的 pavasa3 DNA 為模板向內進行增幅…………… 37 七、將得到的 5’ 端延展的 pavasa3 DNA 片段進行次選殖……………37 拾、利用原位雜合法觀察 pavasa3 mRNA 於豌豆蚜微卵管之表現………37 一、探針載體之建構……………………………………37 二、探針之製備…………………………………………38 三、豌豆蚜微卵管之固定、脫水 (dehydration) 與復水 (rehydration)…38 四、豌豆蚜微卵管與探針之雜合反應…………………39 五、豌豆蚜微卵管與探針雜合後之酵素受質呈色………39 六、豌豆蚜微卵管進行辨識細胞核與細胞骨架之螢光抗體結合………40 拾壹、豌豆蚜體細胞與卵巢細胞 pavasa3 mRNA 表現量比較……………40 一、一般 PCR 定量法………………………………… 40 二、Real-time PCR……………………………………41 拾貳、基本實驗材料之製備與基礎操作……………………41 一、基因體 DNA 的萃取……………………………… 41 二、PCR 產物去除小片段 DNA 以減少黏合至載體之干擾…………42 三、PCR 產物利用電泳切膠以收取目標片段……………42 四、DNA 沉澱…………………………………………… 42 五、接合反應………………………………………………43 六、質體轉形至不同品系之菌體培養……………………43 七、質體 DNA 的萃取………………………………………43 八、RNA 的萃取與 DNaseI 作用…………………………44 九、勝任細胞 C41 之製備……………………………… 45 拾參、各項實驗中使用之引子序列列表………………………46 參考文獻……………………………………………………………48 附圖…………………………………………………………………53 附錄…………………………………………………………………88 | |
| dc.language.iso | zh-TW | |
| dc.title | vasa-like基因在孤雌生殖豌豆蚜發育過程之表現解析 | zh_TW |
| dc.title | Developmental characterization of vasa-like genes in the parthenogenetic pea aphids Acyrthosiphon pisum | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王重雄(Chung-Hsiung Wang),黃火鍊(Fore-Lien Huang),張俊英(Tschining Chang),楊健志(Chien-Chih Yang) | |
| dc.subject.keyword | 豌豆蚜,胚胎發育,生殖細胞, | zh_TW |
| dc.subject.keyword | vasa,pea aphid,Acyrthosiphon pisum,germ cells,germline marker, | en |
| dc.relation.page | 98 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-27 | |
| 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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