【作者】 王睿琪；

【导师】 陈斌； 唐中林；

【作者基本信息】 湖南农业大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 microRNAs(miRNAs)是一类长度18-22(nt)非编码的单链小分子RNA,在动物肌肉发育中起重要调控作用。它通过与靶mRNA的非编码区(3UTR)互补结合,导致mRNA降解或阻断mRNA翻译。研究表明miRNA调控大约30%甚至更多的基因表达,这为解释基因表达和某些相关疾病的分子机理开启了新的途径。在猪的生长发育中,miRNAs对肌肉生长发育中起重要调控作用。研究miRNA对猪骨骼肌生长发育的调控,有利于揭示产肉性状分子机制,为猪的遗传改良提供理论基础。本研究筛选肌肉特异性microRNAs:miR-1a、miR-133b和miR-206a,运用荧光定量方法对其时空表达进行研究。证实在本地猪种和外来猪种孕期母猪各组织的肌肉中miR-1a、miR-133b和miR-206a表达丰富。绘制了外来猪种和本地猪种猪种生长发育不同时期的背最长肌中miRNA的表达图谱。通过生物信息学方法预测三个肌肉特异性miRNAs的潜在靶基因。1.在通城猪不同的组织中,肌肉特异性的mir-1a在肌肉和心脏高丰度表达,在子宫、肾脏、脾脏中低丰度表达；mir-133b在肌肉和心脏都高丰度表达；mir-206a只在肌肉高丰度表达。在长白母猪不同组织中,miR-1a主要在舌肌,背最长肌和腿肌中表达；miR-133b主要是在心肌、舌肌、背最长肌和腿肌中高表达；而miR-206a在舌肌、背最长肌和腿肌中表达丰富。2.在不同时期的背最长肌中,通城猪从胚胎期E33到出生后的D120天miR-1a都有不同程度的表达,出生前低于出生后的表达量。整个发育阶段呈现出四个阶段的差异表达：出生前的胚胎期70天之前表达为低丰度且较为均一,胚胎期E70至E100,出生前E105至出生后的D30时期,出生后D40至D120时期呈上升趋势。长白猪的miR-1则出现了较为明显的两个波段性的表达(E33-E80E90-D30)。3.通城和长白猪的miR-133b出生前胚胎期的骨骼肌中表达丰度都较低,出生后两猪种背最长肌中mir-133b的表达量持续高丰度。出生前的高表达时期通城猪出现在E85天,长白出现在E80天,两猪种都在一个较为相近的时间段。4通城和长白猪整个发育期的背最长肌中都检测出miR-206a的表达,在出生前胚胎期E33都有较高表达,尤其是长白猪的表达量很高。两个猪种的低丰度表达时期分别出现在了E40和E65,而通城猪的高表达时期在出生前的E100,长白出现在了出生后的D9,两猪种都在D100天的时候出现了一个高丰度的表达时期。通过PicTar和TargetScan预测发现miR-1调控肌动蛋白结合蛋白1C(CORO1C)间隙连接蛋白(connexin 43) (GJA1), PFTAIRE蛋白激酶1 (PFTK1); miR-133b调控脂肪瘤高迁移率蛋白IC融合蛋白(LHFP); miR-206a调控端锚聚合酶(TNKS2)。以上结果表明,肌肉特异性miR-1a、miR-133b、miR-206a对本地猪种(通城猪)和外来猪种(长白猪)的肌肉发育都有着一定的调控作用。更多还原

【Abstract】 MicroRNA is a kind of 18-22nt non-coding single RNA, which plays an important role in regulation of muscle development in animal. Mature microRNA leads to degradation or blocking translation of mRNA through bind to 3 UTR (Untranslated Regions). MicroRNAs regulate expression more than 30% of gene. MiRNA opens a new way for gene expression and molecular mechanism of certain disease.MiRNAs conduct regulation of muscle growth evolution conservative network in swine.It contribute to reveal the molecular mechanism of meat traits and provide the theory basis of genetic improvement of pig through researching on miRNA regulation of skeletal muscle growth.This study carried out expression analyis of miR-la miR-133b and miR-206a associated with muscle development based on multipe tissue and developmental muscle using fluorescence relative quantitative methods. It is proved that miR-1a、miR-133b、miR-206a are mainly high-expressed in muscles of obesity-type and lean-type pigs and less in other tissues.It also depicted profiles of longissimus dorsi muscle in different growth time of lean-type and obesity-type pigs and forecasted potential target genes of these miRNAs through bioinformatics approaches.1.Muscle-specific mir-la were abundant in longissimus dorsi muscle and heart, low abundance expression in the uterus, kidney, spleen; mir-133b was highly expressed in heart and longissimus dorsi muscle; Mir-206a only expressed in muscle in Tongcheng pig.MiR-1a and MiR-206a mainly expressed in tongue, longissimus dorsi muscle and crureus, MiR-133b mainly expressed in heart, tongue, longissimus dorsi muscle and crureus in Landrace.2. MiR-1a has different expression level of longissimus dorsi muscle from embryos E33 to D120 after birth of Tongcheng pig and the expression of it is higher in the postnatal than prenatal. It appeared four stages of different expression in the whole development:MiR-la was low abundance before embryo E70 and it is ascended in E70-E100, E105-D30, D40-D120 in Tongcheng pig.MiR-la has a relatively obvious two wave bands of expression:E33-E80 and E90-D30 in Landrace.3.MiR-133b are low abundance in embryo and high abundance in postnatal skeletal muscle of Tongcheng and Landrace.The highest expression stage emerged in a quite close time that E85 in Tongcheng pig and E80 in Landrace.4. MiR-206a was detected in longissimus dorsi muscle in the whole developmental phase of Tongcheng and Landrace. It expressed highly in prenatal E33, especially in Landrace. MiR-206a was lowest abundance in E40 of Tongcheng pig and E65 of Landrace while highest in E100 of Tongcheng and D9 of Landrace.MiR-206a was expressed high in postnatal D100 in two breeds.It procasted that miR-1 regulate CORO1C,GJA1 and PFTK1;miR-133b regulate LHFP;miR-206a regulate TNKS2 through PicTar and TargetScan.The results show that miR-1a、miR-133b、miR-206a have obvious regulation roles in muscle development of obesity-type local pig and lean-type foreign pigs.更多还原