本文選題：鴨 + miRNA-1��； 參考：《揚州大學》2017年碩士論文

【摘要】：microRNAs(miRNAs)是一類非編碼RNA,短單鏈,長度約22 bp,且在進化過程中高度保守。miRNAs在單細胞和多細胞生物中廣泛存在,在轉錄后水平對基因表達進行調控�，F有研究表明miRNA-1和miRNA-133來源于同一對雙順反子(Bicistronic pairs),并對骨骼肌的發(fā)生發(fā)育有重要的調控作用,而關于鴨miRNA-1和miRNA-133對骨骼肌發(fā)育的研究還尚未見有報道。本實驗以體型大小不同的櫻桃谷鴨和莆田黑鴨(白羽系)作為實驗對象,通過實時熒光定量技術構建鴨miRNA-1和miRNA-133的組織表達譜和發(fā)育性表達譜;并通過轉染miRNA的模擬物或抑制物,采用雙熒光素酶報告基因系統對鴨miRNA-1和miRNA-133的功能進行初步探討,以了解其在鴨骨骼肌發(fā)育中的調控作用。主要研究結果如下:1.為探明櫻桃谷鴨和莆田黑鴨(白羽系)的肌肉生長發(fā)育與肌纖維發(fā)育規(guī)律,分別對38、42、45、49、56等不同日齡胸肌重、腿肌重、肌纖維面積等表型進行了測定。結果顯示,櫻桃谷鴨42日齡胸肌重達205 g,腿肌重達238 g,其肌纖維面積分別是5835 μm2和12406μm2,而莆田黑鴨(白羽系)42日齡胸肌重僅為129g,腿肌重175g,其肌纖維面積分別是926 μm2和4089 μm2,表明櫻桃谷鴨與莆田黑鴨(白羽系)在胸肌、腿肌發(fā)育和肌纖維發(fā)育等方面差異顯著。2.為構建鴨miRNA-1和miRNA-133的組織表達譜和發(fā)育性表達譜,本文通過實時熒光定量檢測櫻桃谷鴨和莆田黑鴨(白羽系)的miRNA-1和miRNA-133表達量。檢測結果顯示,miRNA-1和miRNA-133在心肌、胸肌和腿肌等肌肉中呈特異性表達。同時檢測胚胎期和早期生長發(fā)育過程中的肌肉組織miRNA的表達量,發(fā)現鴨胸肌和腿肌miRNA-1和miRNA-133的表達呈現出類似的變化趨勢,即在胚胎期后期miRNA表達急劇上升,而在整個生長發(fā)育期miRNA的表達基本恒定。櫻桃谷鴨中表達量分別在胚胎期28天和生長期42天達到峰值,且在這兩個時間點,櫻桃谷鴨的表達量顯著高于莆田黑鴨(P0.05)。3.為探明鴨miRNA-1和miRNA-133對骨骼肌發(fā)育的作用,將miRNA-1 mimic或inhibitor和miRNA-133 mimic或inhibitor轉染至鴨成肌細胞,實時熒光定量檢測結果顯示,miRNA mimic或inhibitor可有效促進或抑制其表達;且過表達miRNA-1可促進相鄰成肌細胞相互融合,而過表達miRNA-133細胞融合現象很少;CCK-8細胞增殖檢測結果表明,降低miRNA-1表達可促進成肌細胞增殖,而降低miRNA-133則可抑制成肌細胞增殖;成肌細胞分化標記標志基因表達檢測結果顯示,轉染miRNA-1 mimic,分化標記標志基因MEF2d、Myod表達量顯著地上升,而轉染miRNA-133 inhibitor,MEF2d、Myod基因的表達量顯著地上升。以上結果充分說明miRNA-1可促進鴨成肌細胞分化,miRNA-133可促進成肌細胞增殖。4.為進一步闡明miRNA-1和miRNA-133在鴨骨骼肌發(fā)育中的作用機制,通過文獻檢索和生物信息學預測,確定miRNA-1和miRNA-133的候選靶基因。實時熒光定量檢測轉染miRNA的模擬物和抑制物之后候選靶基因的表達量,結果顯示miRNA-1可顯著降低其候選靶基因HDAC4的表達(P0.01),miRNA-133也可降低其候選靶基因SRF、TGFBR1的表達(P0.01)。雙熒光素酶報告系統進一步檢測結果表明,miRNA-1可抑制pGL-Basic-HDAC4熒光素酶報告基因活性;而miRNA-133并未降低pGL-Basic-SRF,pGL-Basic-TGFBR1熒光表達強度。因此,鴨miRNA-1可通過靶向HDAC4促進鴨成肌細胞分化;而miRNA-133可影響SRF、TGFBR1的表達,并促進鴨成肌細胞增殖。
[Abstract]:MicroRNAs (miRNAs) is a class of non coded RNA, short single chain, length about 22 BP, and highly conservative.MiRNAs is widely distributed in single and multicellular organisms during evolution and regulates gene expression at post transcriptional level. Existing studies show that miRNA-1 and miRNA-133 originate from the same pair of CIS counter (Bicistronic pairs) and to skeletal muscle. The development of miRNA-1 and miRNA-133 on skeletal muscle development has not yet been reported. In this experiment, the tissue expression profiles and development of duck miRNA-1 and miRNA-133 were constructed by real time fluorescence quantitative technique with different sizes of Cherry Valley ducks and Putian black ducks (white feather system). The function of duck miRNA-1 and miRNA-133 was preliminarily discussed by the double luciferase reporter gene system by transfection of miRNA mimics or inhibitors. The main results were as follows: 1. the development and development of the muscle and development of Cherry Valley Duck and Putian black duck (white feather) were the main results. The development regularity of muscle fiber was measured on the chest muscle weight, leg muscle weight and muscle fiber area of 38,42,45,49,56, respectively. The results showed that the 42 day old breast muscle of Cherry Valley ducks was 205 g and the leg muscles weighed 238 g, and the muscle fiber area was 5835 m2 and 12406 Mu M2 respectively, while the 42 day old chest muscle weight of Putian black duck (white feather system) was only 129G, leg muscles. The muscle fiber area of 175g was 926 Mu m2 and 4089 M2 respectively, indicating that cherry valley duck and Putian black duck (white feather system) were significantly different in the breast muscle, leg muscle development and muscle fiber development..2. was the tissue expression spectrum and developmental expression profile of miRNA-1 and miRNA-133 in ducks, and the real time fluorescence quantitative detection of Cherry Valley Duck and Putian black duck ( The expression of miRNA-1 and miRNA-133 in the white feather system. The results showed that miRNA-1 and miRNA-133 were specifically expressed in the muscles of the myocardium, the chest muscle and the leg muscles. Meanwhile, the expression of miRNA in the muscle tissue of the embryo and early growth and development was detected, and the expression of miRNA-1 and miRNA-133 in the breast and leg muscles of the duck showed a similar change. The expression of miRNA increased rapidly at the end of the embryo period, while the expression of miRNA in the whole growth period was basically constant. The expression of Cherry Valley ducks reached a peak at 28 days in the embryo period and 42 days in the growth period, and the expression of Cherry Valley ducks was significantly higher than that of Putian black duck (P0.05).3. as the miRNA-1 and miRNA-133 of the ducks. The role of skeletal muscle development is to transfect miRNA-1 mimic or inhibitor and miRNA-133 mimic or inhibitor into duck myoblasts. Real-time fluorescence quantitative detection results show that miRNA mimic or inhibitor can effectively promote or inhibit its expression; and overexpression miRNA-1 can promote adjacent myoblasts fusion, and overexpression miRNA-133 cell fusion now The results of CCK-8 cell proliferation detection showed that the reduction of miRNA-1 expression could promote the proliferation of myoblast, while the reduction of miRNA-133 could inhibit the proliferation of myoblast. The detection results of the marker gene expression of myoblast differentiation showed that miRNA-1 mimic, differentiation marker gene MEF2d, Myod expression increased significantly, and miRNA-13 was transfected to miRNA-13. The expression of 3 inhibitor, MEF2d and Myod genes increased significantly. The above results fully indicate that miRNA-1 can promote the differentiation of duck adult myoblast. MiRNA-133 can promote the proliferation of myoblast by.4. to further clarify the mechanism of miRNA-1 and miRNA-133 in the development of skeletal muscle in ducks. By literature retrieval and bioinformatics prediction, miRNA-1 and miRNA- are determined. 133 candidate target gene. Real-time fluorescence quantitative detection of the expression of candidate target gene after transfection of miRNA mimics and inhibitors. The results show that miRNA-1 can significantly reduce the expression of the candidate target gene HDAC4 (P0.01), miRNA-133 also reduces the candidate target gene SRF, TGFBR1's apparent (P0.01). Double luciferase reporter system is further detected. The results showed that miRNA-1 could inhibit the activity of pGL-Basic-HDAC4 luciferase reporter gene, while miRNA-133 did not reduce pGL-Basic-SRF and pGL-Basic-TGFBR1 fluorescence intensity. Therefore, ducks miRNA-1 can promote the differentiation of duck myoblasts by targeting HDAC4, and miRNA-133 can affect the expression of SRF, TGFBR1, and promote the proliferation of duck myoblasts.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S834

【參考文獻】

相關期刊論文 前4條

1 劉長召;王玲;陳文江;;大鼠心肌細胞缺氧后miR-133a-3p的表達及其下游靶基因預測[J];局解手術學雜志;2016年11期

2 陶志云;朱春紅;姬改革;徐文娟;單艷菊;李慧芳;;鴨胚骨骼肌成肌細胞中MyoD1和Myf5基因的表達與分析[J];福建農林大學學報(自然科學版);2014年03期

3 弓賀煒;李波;李文斌;李剛;梁炳生;;miRNA-1和miRNA-133過表達對L6細胞增殖與分化的影響[J];中華細胞與干細胞雜志(電子版);2014年02期

4 劉昒;趙琴平;董惠芬;蔣明森;;TGF-β信號傳導通路及其生物學功能[J];中國病原生物學雜志;2014年01期

相關博士學位論文 前3條

1 吳瑛;蛋鴨產業(yè)組織行為分析與組織創(chuàng)新研究[D];華中農業(yè)大學;2013年

2 朱文奇;高郵鴨、金定鴨胚胎期骨骼肌生長發(fā)育的研究[D];揚州大學;2014年

3 羅月球;1. MiR-133a，miR-1，，miR-206誘導C2C12細胞成肌前體細胞及miR-133a靶基因Fox12鑒定的研究 2. IL-28B多態(tài)性位點及血清維生素D水平在HCV感染病人中的相關性分析研究[D];浙江大學;2015年

相關碩士學位論文 前2條

1 王麗娟;MiR-1和miR-133在山羊骨骼肌組織和細胞中的表達規(guī)律及功能研究[D];安徽農業(yè)大學;2014年

2 王睿琪;豬肌肉生長相關microRNA-1a，133b和206a的表達分析[D];湖南農業(yè)大學;2011年

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