【作者】 顾丽红；

【导师】 孙世铎；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2015， 博士

【摘要】 Z型北京鸭配套系是以国内的北京鸭为基础培育的新品系,具有瘦肉率和饲料转化效率高的特点,该配套系的育成为满足不同消费形式以及研究鸭骨骼肌发育提供了良好材料。表型的形成是控制该性状的基因和环境互作的结果,其中基因是表型形成的遗传基础。因此,分离鉴定控制性状的基因并对基因表达起重要调控作用的mi RNAs进行鉴定分析是探索鸭骨骼肌发育机制的有效途径。鉴于此,本研究应用高通量测序技术和生物信息学技术研究了北京鸭胚胎期胸肌内基因和mi RNAs的表达模式与特征,探索了基因与mi RNAs的互作关系,同时应用RNA干扰和过表达技术研究了部分新mi RNAs的功能。得到以下研究结果:(1)孵化第19天鸭胚(E19)是胚胎期北京鸭胸肌发育最快的时期,是北京鸭胚胸肌发育的关键点;(2)胚胎期北京鸭胸肌内差异表达基因分析和差异表达基因的KEGG pathway分析表明:E27时北京鸭胸肌发育状态与E13和E19的胸肌发育状态有较大差异,E13北京鸭胸肌内已经初具脂肪和脂溶性物质的调节能力;E19北京鸭胸肌内调节脂肪发育通路显著富集,E27北京鸭胸肌内代谢相关通路占绝对优势;(3)胚胎期北京鸭胸肌内mi RNAs表达模式研究鉴定出359个已知mi RNAs和23个新mi RNAs;其中应用q RT-PCR技术验证的15个已知mi RNAs表达模式可以分为三类:在E19胸肌内高表达、在E19低表达并随鸭胚胸肌的生长其表达量升高;23个新mi RNAs中只有novel-mir-8和novel-mir-14在胸肌中高表达,进一步分析及实验验证发现novel-mir-8可能是胚胎期胸肌发育的抑制者,而novel-mir-14可能是胚胎期胸肌发育的促进者;(4)显著差异的mi RNA和m RNA关联分析显示:E13-E19中43个差异显著的mi RNA关联22个差异显著的m RNA,这些差异显著的m RNA主要与肌肉的发生、发育和分化相关;E19-E27中48个显著差异的mi RNA关联141个显著差异的m RNA,差异显著的m RNA与细胞周期和DNA复制关系密切;(5)应用超表达和干扰技术研究了novel-mi R-6、novel-mi R-17和novel-mi R-20对成肌细胞分化的影响,结果显示novel-mi R-6对成肌分化的作用尚难以定论;novel-mi R-17和novel-mi R-20可能具有抑制成肌分化的作用。本研究能够从基因水平及mi RNAs对基因的影响等方面较深入地探讨北京鸭胚胎期骨骼肌发育的机制,为更深入地开展北京鸭分子育种提供理论基础和依据,同时能够为其它畜禽骨骼肌发育研究提供参考。更多还原

【Abstract】 The lean line of Z-type Beijing duck with higher lean meat ratio and higher feed conversion efficiency was successfully bred based on the initial Beijing duck. The lean line of Beijing duck can meet different consumer demand and can provide an ideal animal model for the exploration of skeletal muscle development mechanism. The formation of phenotype is the interaction results of genes and environment, the genes are genetic basis of the phenotype formation. Therefore, the isolation and identification of genes controlled the related trait and mi RNAs regulated the above genes are an effective way in exploration of the mechanism of skeletal muscle development.Based on the fact above, we identified the expression patterns and characteristics of the genes and mi RNAs in embryonic breast muscle of Beijing duck and their relationship between the identified genes and mi RNAs above by using high throughput sequencing and bioinformatics technology analysis. In addition, the effect of some novel mi RNAs predicted through our mi RNAs sequencing data on the differentiation of myogenesis was explored usng overexpression and RNAi technology. The results were listed as follow.(1) E19 is the fastest point of breast muscle development and that E19 is also the crucial transition for Pekin duck pectoral muscle development during embryonic stage.(2) The analysis of differentially expressed genes in embryonic breast muscle of Beijing duck and the KEGG pathway analysis of these genes showed that there were huge difference between the breast muscle development state of E27 and that of E13 and E19. There was an initial regulation for fat and fat-soluble substance in E13 breast muscle of Beijing duck, the related pathways of fat development were significantly enriched in E19 breast muscle of Beijing duck, and the related pathways of metabolism were dominant in E27 breast muscle of Beijing duck.(3) Totally, 359 known mi RNAs and 23 novel mi RNAs were identified in E13, E19 and E27 breast muscle of Beijing duck. The expression patterns of 15 muscle-related known mi RNAs, whose expression were validated by using q RT-PCR, can be divided into three types: the mi RNAs with their expression increasing from E11 to E27, the mi RNAs with their expression reached to the bottom at E19 and the mi RNAs with their expression peaked at E19. Among the 23 novel mi RNAs, only novel-mir-8 and novel-mir-14 highly expressed in embryonic breast muscle of Beijing duck, and the subsequent functional validation study indicated that novel-mir-8 was a potential inhibitor and novel-mir-14 was a potential promotor.(4) The significantly association analysis between mi RNAs and m RNA were carried out. In group of E13-E19, 43 differetially expressed mi RNAs and 22 differetially expressed m RNAs were surported each other and the 22 differetially expressed m RNAs were mainly associated with the myogenesis, development and differentiation of skeletal muscle. In group of E19-E27, 48 differetially expressed mi RNAs and 141 differetially expressed m RNA were surported each other. The differetially expressed genes above were involved in DNA replication, myogenic, muscle development and cell cycle biological processes.(5) The functional exploration of novel-mi R-6, novel-mi R-17 and novel-mi R-20 on the myogenesis differentiation of myoblasts cell was performed by using overexpression and inference technology. The results showed that novel-mi R-17 and novel-mi R-20 were both the inhibitors of myogenesis differentiation of myoblasts cell, but the function of novel-mi R-6 was not clear.The results of this research could provide a method to explore the mechanism of embryonic breast muscle development of Beijing duck from genes and mi RNAs level, could provide theoretical basis and foundation for molecular breeding of Beijing duck, and could also provide a model for the study of skeletal muscle development of other animals.更多还原