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研究生:顏呈峰
研究生(外文):Cheng-Feng Yen
論文名稱:菜鴨脂質代謝相關基因之選殖與表現
論文名稱(外文):Cloning and expression of genes associated with lipid metabolism in Tsaiya ducks
指導教授:姜延年姜延年引用關係
指導教授(外文):Yan-Nian Jiang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:畜產學研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:79
中文關鍵詞:脂質代謝菜鴨
外文關鍵詞:lipid metabolismTsaiya duck
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菜鴨(Anas platyrhynchos var. domestica)為台灣主要產蛋鴨之品種,產蛋率非常高。欲維持高產蛋率則需要有極高的脂質新生合成作用以提供蛋黃堆積所需。固醇調節因子結合蛋白(sterol regulatory element binding pretins, SREBPs)可能參與調控肝臟脂質生成相關基因的表現,而脂質生成的能力則直接影響蛋黃脂質的堆積。在哺乳動物中,SREBP1與2能調控脂肪酸與膽固醇合成相關基因,但在家禽方面,SREBP1與2基因的表現以及其與脂質代謝的相關性則未見研究。在產蛋家禽脂質利用扮演重要角色的極低密度衍脂蛋白-II(very low density apolipoprotein-II, apoVLDL-II)在菜鴨也未被研究。本實驗之目的在選殖菜鴨SREBP 1、SREBP2、脂肪酸合成酶(FAS)、3-羥基-3-甲基戊二酸輔酶A還原酶(HMG-CoA reductase)及apoVLDL-II五個與脂質新生合成及利用相關基因,並探討其在菜鴨體內組織及產蛋前後表現,以期增進對產蛋菜鴨脂質代謝相關基因表現的了解。
試驗以30隻菜鴨為材料,15隻在產蛋前(18週齡)犧牲,另外15隻則在產蛋高峰時(整群產蛋率達80%)犧牲。菜鴨在犧牲後立即採取腹部脂肪、心臟、肝臟、骨骼肌及卵巢組織,並儘速以液態氮冷凍隨後貯存於-80℃。萃取組織RNA後進行反轉錄聚合酶連鎖反應(RT-PCR),選殖出菜鴨SREBP 1、SREBP2、FAS、HMG-CoA reductase基因片段,此外並在菜鴨肝臟全長cDNA基因庫中篩選出菜鴨apoVLDL-II之全長cDNA。經序列分析後發現菜鴨SREBP1、SREBP2、FAS及HMG-CoA reductase基因與蛋雞有高度之同源性,顯示兩物種間有高度的演化遺傳關聯性。而菜鴨之apoVLDL-II基因全長與蛋雞和鵪鶉的相似度分別為87%與82%。以北方吸漬分析法分析各基因在菜鴨的組織中表現結果顯示SREBP1 mRNA可以在脂肪組織、心肌、骨骼肌、肝臟及卵巢中表現。SREBP2 mRNA濃度在肝臟與卵巢中較其他組織都為高。FAS與HMG-CoA reductase mRNA在肝臟中大量表現而其他組織則較少。apoVLDL-II mRNA則只在肝臟中測得,其他組織檢測不到。試驗中亦發現,產蛋高峰與產蛋前菜鴨肝臟中SREBP1、SREBP2及FAS mRNA表現量並無明顯差異。然而,產蛋高峰菜鴨肝臟中HMG-CoA reductase 與apoVLDL-II mRNA的表現量顯著高於產蛋前菜鴨。試驗結果顯示產蛋可能會對特定脂質代謝的途徑造成影響,尤其會影響與蛋黃組成分堆積相關的apoVLDL-II及HMG-CoA reductase。而在基因表現差異實驗中發現,菜鴨在開始產蛋後肝臟內部分與卵黃形成及脂質代謝利用相關基因會大量表現,尤其是vitellogenin與apoVLDL-II等與卵黃脂質堆積有密切關係的基因。這些選殖到的基因仍需經進一步的確認其在產蛋前後表現上的差異,而試驗中另亦選殖到釵h新穎的基因,其參與產蛋機能的角色亦待進一步的探討。
Tsaiya duck is the major egg-laying duck in Taiwan that has high egg production rate. To support egg yolk lipid deposition for Tyaiya duck needs a high lipid generation machinery to support the egg yolk lipid accumulation. In mammals, sterol regulatory element binding protein 1 (SREBP1) drives the expression of several lipogenic genes whereas SREBP2 dictates the expression of every gene involved in cholesterogenesis. The expression of these two genes and their target genes is an indication of the capability of hepatic lipid metabolism. The ability of adequate lipid formation is an assurance for yolk lipid deposition. With much of the emphasis been on mammalian species, the expression of SREBP1 and SREBP2 and their relation with avian lipid metabolism has not been well characterized. Very low density apolipoprotein-II (apoVLDL-II), a protein only expressed in female birds, plays an important role on lipid transportation from liver to ovary, and has not been studied for Tsaiya duck. Our purposes are to clone the gene fragments of Tsaiya duck SREBP1, SREBP2, FAS, HMG CoA reductase, and apoVLDL-II, the genes associated with lipid metabolism and to determine tissue distribution of these genes. The effects of egg-laying on the mRNA concentrations of SREBP1, SREBP2, FAS, HMG CoA reductase, and ApoVLDL-II in liver of Tsaiya ducks were also studied.
Fifteen ducks right before the first egg was laid (18-wk old) and 15 ducks from the same population at a egg production rate of 80 % were sacrificed. Adipose tissue, cardiac muscle, skeletal muscle, liver, and ovary were quickly dissected, frozen in liquid nitrogen, and stored at –80 ℃. The total RNA was extracted and used to clone the genes fragments of SREBP1, SREBP2, FAS and HMG-CoA reductase by RT-PCR. We also cloned the Tsaiya duck apoVLDL-II full length cDNA from a Taiya duck liver cDNA library。The data showed that Tsaiya duck SREBP1, SREBP2, FAS and HMG-CoA reductase were highly homologous to that of chicken, indicating the genetic relationship between those two species was close. The tissue distribution of this five genes were determined by northern analysis. The data showed that Tsaiya duck SREBP1 mRNA was expressed in adipose tissue, cardiac muscle, skeletal muscle, liver, and ovary. The SREBP2 mRNA concentration was high in the liver and ovary. The FAS and HMG-CoA reductase mRNA were highly expressed in the liver and to a lesser extend in other tissues. The apoVLDL-II mRNA was specificly expressed in the liver. The differences of mRNA concentrations of SREBP1, SREBP2, FAS in the livers of laying and pre-lay ducks were not significant. However, the concentrations of hepatic HMG-CoA redutase and ApoVLDL-II mRNA were higher in the laying ducks than that of pre-lay duck. The data suggest that laying may affect particular aspect of lipid metabolism, especially biochemical pathways that apoVLDL-II and HMG-CoA reductase were involved. The data of suppression subtractive hybridization showed that the expression of genes such as vittellogenin and apoVLDL-II, genes asociated with yolk formation and lipid metabolism, was increased after laying. The expressed differentiation of the genes we cloned were needed more confirmation. We also cloned many novel genes and their function attend at egg-laying were needed confirmation. The differentially expressed genes need further confirmation. The functions of those novel genes discovered in the suppression subtractive hybridization need to be studied.
目錄 I
表次 II
圖次 III
摘要 1
前言 3
文獻檢討
一、脂質代謝 4
二、脂質代謝的調節 11
三、產蛋家禽對脂質的利用 20
材料與方法 25
結果與討論 37
結論 63
參考文獻 64
英文摘要 78
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