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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張繼堯(Chi-Yao Chang) | |
| dc.contributor.author | Chun-Ping Wang | en |
| dc.contributor.author | 王俊平 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:22:35Z | - |
| dc.date.available | 2010-08-20 | |
| dc.date.copyright | 2009-08-20 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-18 | |
| dc.identifier.citation | 山元孝吉，1971。袋形動物、輪蟲綱。新日本動物圖鑑上冊，岡田要編 三版404-423，北隆館。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43507 | - |
| dc.description.abstract | 石斑魚(Grouper)育苗前期，以輪蟲(Rotifer, Brachionus plicatilis)為主要餌料生物，然而輪蟲營養價值不穩定，魚苗攝食後往往造成n-3高度不飽和脂肪酸(highly unsaturated fatty acid, HUFA)缺乏，進而增加魚體畸形和高死亡率。本研究以營養基因體學觀點，探討輪蟲營養強化後對點帶石斑(Epinephelus coioides)營養組成和骨骼發育之影響，並利用微陣列生物晶片(microarray biochip)探討表現差異之基因群，以建立健康石斑魚苗外表型與基因型關聯之基因標誌，作為營養需求與胚胎發育研究的基礎。本研究有兩組輪蟲滋養液配方，甲組為超級熊克(Super DC Selco)，乙組為鱈魚乳化油，比照未滋養輪蟲之脂肪酸組成，超級熊克滋養輪蟲後其體內之粗脂質增加0.79 %，鱈魚乳化油滋養之粗脂質增加1.72 % ，而魚苗攝食滋養輪蟲比較攝食未滋養者，超級熊克滋養輪蟲餵飼之魚苗粗脂質增加0.48 %，鱈魚乳化油組粗脂質增加0.39 %。在花生四烯酸(arachidonic acid, ARA)組成上，以油脂滋養輪蟲投餵魚苗組較未滋養組含量減少，然而二十碳五烯酸(eicosapentaenoic acid, EPA)、二十二碳六烯酸(docosahexaenoic acid, DHA)及n-3 HUFA之蓄積量皆有顯著增加，超級熊克滋養輪蟲餵飼魚苗之n-3 HUFA達8.7 %，鱈魚乳化油組達8.9 %。輪蟲和魚苗間之脂肪酸轉換比例也呈現正向關係，魚苗不管攝食未滋養或甲乙組滋養輪蟲，其體內DHA和EPA比值皆大於1，其中超級熊克組更高達4以上。過氧化體增生活化受體 γ (peroxisome proliferators-activated receptor γ, PPAR γ)是調控脂肪代謝相關基因表現的重要轉錄因子，以RT-PCR比較滋養組和未滋養組全魚體之PPAR γ表現量，發現各組別間並沒有顯著差異，推論未滋養或滋養輪蟲之營養值已頗高，皆可維持其PPAR γ的表現量。由石斑魚胚胎發育期cDNA基因庫構築之微陣列生物晶片，分析超級熊克和乳化鱈魚油滋養組比較於未滋養組輪蟲餵食後之魚苗基因表現變化，發現共交集1.5倍以上促進表現(up-regulation)之基因群有37個，1.5倍以上抑制表現(down-regulation)之基因群有27個，進一步利用NCBI基因庫比對得知，促進表現之差異性基因群有骨骼及肌肉發育相關之結構蛋白質，如： myosin light chain 3、actin alpha skeletal muscle、muscle creatine kinase等，也有生理代謝相關酵素，如：chymotrypsinogen 2、pyruvate kinase、pancreatic carboxypeptidase B1、enolase 3、apolipoprotein A和hypothetical protein LOC等。抑制表現之差異性基因群有能量代謝相關之蛋白質：cytochrome c oxidase subunit III；結構蛋白質：alpha 2 type I collagen；DNA複製之關鍵酵素：checkpoint kinase 1；激活素受體：activin receptor IIA等。本研究結果顯示，點帶石斑魚苗餵食n-3 HUFA滋養後之輪蟲，可增加魚體粗脂質、EPA、DHA及n-3 HUFA含量，DHA/ EPA比值亦符合營養指標。透過微陣列生物晶片分析，可推論餌料生物營養組成與點帶石斑魚苗發育之關聯性。 | zh_TW |
| dc.description.abstract | In the early stage of grouper larvae culture, Rotifer (Brachionus pliicatilis) is the major living feed. However, the nutrition of rotifer is not stable, which leads to the lack of n-3 highly unsaturated fatty acid (HUFA) in larvae and the increase of malformation rate and mortality. In this study, we investigated the effect of rotifer nutrition enrichment on nutrition composition and skeleton development of Epinephelus coioides from the nutrition genomics point of view. We investigated the differentially expressed genes by microarray biochip to establish the gene markers relationship between healthy phenotype and genotype as the basis for nutrition needs and embryo development. There are two rotifer enrichment formulas designed in this study, one is Super DC Selco, the other is cod emulsion oil. After rotifer enrichment, the crude lipid content increases 0.79 % (Super DC Selco), 1.72 % (cod emulsion oil) in rotifer, and 0.48 % (Super DC Selco), 0.39 % (cod emulsion oil) in larvae. When the larvae group fed with rotifer enriched with emulsion, respectively the arachidonic acid (ARA) composition decreased. However, the accumulation amount of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and n-3 highly unsaturated fatty acid (HUFA) were significantly increased. The n-3 HUFA of larvae fed rotifer increased 8.7 % enriched with Super DC Selco, and 8.9 % enriched with cod emulsion oil, respectively. The fatty acid transfer through the food chain between rotifer and larvae was proportional. The DHA/EPA ratio was above 1, no matter the larvae was fed with enriched- or un-enriched- rotifer, and the DHA/EPA ratio even reached 4 for larvae fed with rotifer enriched with Super DC Selco. Peroxisome proliferator-activated receptor γ (PPAR γ) is an important transcription factor in regulating gene expression of lipid metabolism. Compare the expression amount of PPAR γ from grouper larvae in enriched group and un-enriched group by RT-PCR, we found that there is no significant difference between each group, which indicated that the nutrition of un-enriched- or enriched- rotifer has high nutrition already. Using the microarray biochip made from grouper embryonic stage cDNA library to analyze the gene expression profile of larvae fed with rotifer enriched with Super DC Selco or cod emulsion oil or un-enriched rotifer, we found that there were 37 genes with 1.5 fold up-regulation and 27 genes with 1.5 fold down-regulation. Further NCBI blast results showed that the up-regulated genes include skeletal and muscle development related structural proteins：ex myosin light chain 3, actin alpha skeletal muscle and muscle creatine kinase; metabolism related enzymes：ex chymotrypsinogen 2, pyruvate kinase, pancreatic carboxypeptidase B1, enolase 3, apolipoprotein A and hypothetical protein LOC. Whereas down-regulated genes include energy metabolism related protein： cytochrome c oxidase subunit III；structural protein：alpha 2 type I collagen；DNA replication related protein：checkpoint kinase 1 and activin receptor IIA. It is concluded that grouper larvae fed with enriched- rotifer the total n-3 HUFA was increased compare to that fed with un-enriched- rotifer in the composition of crude lipid, EPA, DHA and n-3 HUFA. Therefore microarray technique can establish the nutrition composition of living feed correlation with development of grouper larvae. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:22:35Z (GMT). No. of bitstreams: 1 ntu-98-R96b45026-1.pdf: 1327296 bytes, checksum: c54a977f479fdecc53b87b23c8973ab5 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 中文摘要 …………………………………………………………………………….Ι
英文摘要 …………………………………………………………………………....Ⅲ 表目次 ……………………………………………………………………………....Ⅷ 圖目次 ……………………………………………………………………………....Ⅸ 一、 前言 …………………………………………………………………………1 二、 文獻回顧 ……………………………………………………………………4 2.1 水產種苗培育 ……………………………………………………………4 2.2 石斑魚(Epinephelus spp.) ………………………………………………..4 2.2.1. 石斑魚介紹 ………………………………………………………4 2.2.2 石斑魚繁殖與胚胎發育 …………………………………………5 2.3 餌料生物…………………………………………………………………7 2.3.1 輪蟲介紹 …………………………………………………………7 2.3.2 培育條件及營養價值 ……………………………………………8 2.4 脂肪酸……………………………………………………………………9 2.4.1脂肪酸介紹 ………………………………………………………9 2.4.2 魚類脂肪酸需求 ………………………………………………10 2.4.3 魚類體脂肪分佈…………………………………………………11 2.4.4 脂肪組成對魚類發育之外表型分析……………………………12 2.4.5 脂肪組成對魚類發育之基因型分析……………………………12 2.5 過氧化體增生活化受體(PPAR)之生理特性…………………………14 2.5.1 PPAR的發現 …………………………………………………14 2.5.2 PPAR的種類 …………………………………………………14 2.5.3 PPAR的結構 …………………………………………………15 2.5.4 PPRE…………………………………………………………..16 2.5.5 PPAR之ligands ………………………………………………17 2.5.6 PPAR γ和脂肪生合成 ………………………………………17 2.5.7 魚類PPARs之生理角色 ………………………………………18 三、 材料與方法 ……………………………………………………………….20 3.1 餌料生物培養…………………………………………………………20 3.1.1 輪蟲培養 ……………………………………………………20 3.1.2 藻類培養 ……………………………………………………20 3.1.3 韋因培養液配製………………………………………………21 3.2 輪蟲滋養………………………………………………………………21 3.3 點帶石斑(Epinephelus coioides)魚苗培育 …………………………22 3.4 粗脂質萃取……………………………………………………………22 3.5 脂肪酸萃取……………………………………………………………23 3.5.1 皂化 …………………………………………………………23 3.5.2 甲基酯化 ……………………………………………………23 3.5.3 氣相層析儀(GLC)分析………………………………………24 3.6 透明魚骨骼樣本製作與觀察 ………………………………………24 3.7 全魚體Total RNA之萃取 …………………………………………25 3.8 RNA變性電泳分析 ………………………………………………25 3.9 RNA cleanup純化………………………………………………… 26 3.10 點帶石斑之過氧化體增生活化受體基因選殖……………………27 3.10.1 專一性引子設計……………………………………………27 3.10.2 互補去氧核醣核酸(cDNA)製備……………………………27 3.10.2.1 One-step反轉錄聚合酶鏈鎖反應………………27 3.10.2.2 Two-step聚合酶鏈鎖反應 ……………………28 3.10.2.3 洋菜膠體電泳……………………………………29 3.10.3 載體接合作用………………………………………………29 3.10.4 勝任細胞製備………………………………………………30 3.10.5 轉形作用……………………………………………………30 3.10.6 質體DNA純化 ……………………………………………31 3.10.7 限制酶剪切作用……………………………………………31 3.10.8 DNA定序…………………………………………………32 3.10.9 PPAR γ基因表現量分析…………………………………32 3.11 微陣列生物晶片(Microarray biochip)分析………………………33 3.11.1 點帶石斑魚受精卵至白身期之cDNA基因庫製備………33 3.11.2 魚苗探針(Probe)製備………………………………………33 3.11.2.1 全魚體Total RNA之萃取同3.7……………….33 3.11.2.2 RNA變性電泳分析同3.8…………………….33 3.11.2.3 RNA cleanup純化同3.9………………………33 3.11.2.4 第一股cDNA合成 ……………………………33 3.11.2.5 cDNA coupling dye……………………………34 3.11.2.6 微陣列生物晶片前準備 ………………………34 3.11.2.7 雜合反應(Hybridization)……………………….34 3.11.2.8 差異性基因表現分析 …………………………35 3.11.2.9 候選基因定序分析……………………………35 四、 結果……………………………………………………………………..37 五、 討論…………………………………………………………………….44 六、 參考文獻……………………………………………………………….51 七、 表……………………………………………………………………….61 八、 圖……………………………………………………………………….71 | |
| dc.language.iso | zh-TW | |
| dc.subject | 過氧化體增生活化受體 γ | zh_TW |
| dc.subject | 輪蟲 | zh_TW |
| dc.subject | n-3高度不飽和脂肪酸 | zh_TW |
| dc.subject | 石斑魚 | zh_TW |
| dc.subject | 花生四烯酸 | zh_TW |
| dc.subject | 二十二碳六烯酸 | zh_TW |
| dc.subject | 二十碳五烯酸 | zh_TW |
| dc.subject | PPAR γ | en |
| dc.subject | Grouper | en |
| dc.subject | Rotifer | en |
| dc.subject | n-3 HUFA | en |
| dc.subject | ARA | en |
| dc.subject | EPA | en |
| dc.subject | DHA | en |
| dc.title | 比較不同高度不飽和脂肪酸滋養之輪蟲餵食點帶石斑魚苗之發生與基因表現 | zh_TW |
| dc.title | Comparison of gene expression profile and development of red spotted grouper (Epinephelus coioides) larvae fed with various highly unsaturated fatty acids enriched rotifer | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 廖文亮(Wen-Liang Liao) | |
| dc.contributor.oralexamcommittee | 蘇建國(Jyan-Gwo J. Su) | |
| dc.subject.keyword | 石斑魚,輪蟲,n-3高度不飽和脂肪酸,花生四烯酸,二十碳五烯酸,二十二碳六烯酸,過氧化體增生活化受體 γ, | zh_TW |
| dc.subject.keyword | Grouper,Rotifer,n-3 HUFA,ARA,EPA,DHA,PPAR γ, | en |
| dc.relation.page | 100 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
| 顯示於系所單位： | 漁業科學研究所 | |
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