- 學位論文
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
Comparison of gene expression profile and development of red spotted grouper (Epinephelus coioides) larvae fed with various highly unsaturated fatty acids enriched rotifer
摘要
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。
並列摘要
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.
參考文獻
延伸閱讀
- 高瑞國（2009）。比較橈足類與豐年蝦餵食點帶石斑魚苗之發生與基因表現〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2009.10305
- 謝米柔（2015）。基因轉殖水稻表現石斑魚虹彩病毒主要外鞘蛋白之研究〔碩士論文，國立宜蘭大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0046-2608201511305700
- 卓立超（2014）。探討富含DHA之裂殖壺藻粉取代飼料中魚油對點帶石斑稚魚成長表現、脂肪酸組成及免疫反應之影響〔碩士論文，國立屏東科技大學〕。華藝線上圖書館。https://doi.org/10.6346/NPUST.2014.00185
- 余志儒、許北辰（2015）。錨紋瓢蟲(Lemnia biplagiata)成蟲以不同性比與密度飼育之壽命與卵穫量。植物保護學會會刊，57(4)，71-82。https://doi.org/10.6715/PPB.201512_57(4).0001
- 蔡旻哲（2014）。Effects of dietary different in encapsulated and unencapsulated nature digestive enzymes on growth and immune responses of orange-spotted grouper (Epinephelus coioides)〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2014.01519