【作者】 徐铁山；

【导师】 刘小林； 侯水生；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2014， 博士

【摘要】 北京鸭是世界著名的肉鸭品种，其中” Z型北京鸭瘦肉型配套系”具有饲料转化效率高、瘦肉率高的特点，是研究禽类骨骼肌发育的极好模型，但其骨骼肌发育研究尚不多见。但是，骨骼肌形成与再生的分子机制远非单个调节因子作用的加减，而是所有调节因子相互作用的网络。因此，以大规模的数据分析为基础寻找不同生长阶段北京鸭胸肌发育的差异表达基因及差异表达基因与北京鸭胸肌的关系研究是深度挖掘骨骼肌发育机制的基础。本研究应用抑制消减杂交（SSH）和RNA-seq技术分离鉴定了不同生长阶段北京鸭胸肌发育差异表达基因。分析了经典的骨骼肌发育调控基因（MSTN、IGF1和FOXO3）分子特征、它们在北京鸭胸肌发育过程中的表达变化规律及其与胸肌发育之间的关系。同时，开展了新发现的MUSTN1基因分子特征、时空表达模式及其与北京鸭胸腿肌发育的关系以及北京鸭胸肌内活体转染pcDNA3.1（+）-MUSTN1真核表达载体以探索MUSTN1基因对北京鸭胸肌发育的作用。试验方法及结果如下：1、应用SSH技术分离鉴定不同生长阶段北京鸭胸肌发育差异表达基因应用SSH技术从2周龄和6周龄北京鸭胸肌SSH文库中分离得到257个差异表达基因（165个已知基因和92个未知基因）。多数差异表达基因在6周龄北京鸭胸肌内高表达且与机体的能量代谢和蛋白合成有关。利用qRT-PCR分析了23个差异表达基因在北京鸭2、4、6、8周龄胸肌中的表达模式，通过聚类分析可以将这23个基因分为表达量持续下降、表达量先升高后下降和表达量持续上升等三种类别。2、应用RNA-seq技术分离鉴定不同生长阶段北京鸭胸肌发育的差异表达基因对2、4、6周龄北京鸭胸肌和皮脂组织进行cDNA文库构建及RNA-seq测序与分析，共得到21800万个高质量序列，占原始序列的95.76%。生物信息学分析发现这些高质量序列在鸭子参考基因上随机分布，覆盖了鸭子已注释基因的89.4%，序列的RPKM值与荧光定量值整体趋势一致。相同时期组织间及同一组织不同生长阶段间的差异表达基因数量从几百个到数千个，其中组织间差异表达基因远多于生长阶段间，而皮脂间差异表达基因多于胸肌间。GO和KEGG分析发现，差异表达基因被富集到不同GO条目中，而大量差异表达基因（如IGF1R、IGF1、MSTN、TGFβ3、TGFβ1、TGIF1、FOXO6、FOXO3等）被显著富集到肌肉发育及脂肪沉积的信号通路中，仅MAPK通路就富集了214个差异表达基因。3、肌肉抑制素基因（MSTN）分子特征及其多态性与北京鸭胸肌性状的关联分析应用qRT-PCR技术检测MSTN基因在第1d和2、4、6、8周龄北京鸭胸肌中的表达量发现：从1d到4周龄该基因表达量逐渐升高，但6周龄的表达量显著低于4周龄和8周龄的表达量。分子克隆及生物信息分析发现，北京鸭MSTN基因的CDS区长度为1128bp，其编码蛋白质与多数脊椎动物同源蛋白质具有很高的相似性。MSTN基因具有2个可变剪切体，分别编码375个氨基酸序列和251个氨基酸序列。通过对368只6周龄北京鸭该基因的克隆、SNP扫描、分型及关联分析发现：该基因含有3个SNP位点，其中T129C位点BB基因型鸭子的胸肌厚显著大于AA基因型鸭子的胸肌厚，T952C位点AB基因型鸭子的龙骨长显著长于BB基因型鸭子的龙骨长。4、IGF1和FOXO3表达模式及出雏后北京鸭胸腿肌发育关系研究北京鸭胸腿肌在第2d和2、4、6周龄石蜡切片表明，北京鸭胸肌发育落后于腿肌，其中胸肌在4~6周龄生长最快，而腿肌在2~4周龄生长速度最大。应用qRT-PCR技术检测IGF1和FOXO3在2d和2、4、6周龄胸腿肌中表达情况表明，IGF1分别在第6周和第2周胸肌和腿肌中的表达量最大，而FOXO3分别在第6周和第2周胸肌内表达量最小。5、MUSTN1基因分子特征及其与北京鸭胸肌发育关系研究经过对北京鸭MUSTN1基因cDNA和DNA序列克隆、拼接与分析发现，该基因编码一个含有78个氨基酸的蛋白质，由3个外显子和2个内含子组成，其启动子区包含许多转录因子结合位点。MUSTN1基因在北京鸭胸肌、腿肌中的表达量分别在5周龄和3周龄达到峰值。活体转染pcDNA3.1+MUSTN1表达载体到北京鸭胸肌后，该重组载体能够在北京鸭胸肌中高效表达，同时引起一些骨骼肌发育调控基因表达量的明显变化。综上所述，本研究分离鉴定了北京鸭胸肌发育不同阶段的差异表达基因。对部分经典的骨骼肌发育调控基因及新发现的MUSTN1基因进行了分子特征及其与北京鸭胸肌发育的关系研究。本研究结果将对深入探索北京鸭骨骼肌发育机制提供基础。更多还原

【Abstract】 Pekin duck is a world-famous meat breed. The Z-type lean Pekin duck, one line of Pekinduck, is a ideal model for the study of skeletal muscle development due to its characteristicsof high feed conversion efficiency and high lean meat. However, the study about skeletaldevelopment of Z-type lean Pekin duck is still rare currently. The formation and reparation ofskeletal muscles is much more than the sum of its individual regulators. Therefore, it is thebasis of exploring the mechanism of skeletal muscle development that picking out thedifferentially expressed genes of various developmental stages in Pekin duck breast musclesbased on the analysis of large-scale data and clearing the relationship between thedifferentially expressed genes and muscle development.The differentially expressed genes in Pekin duck breast muscles were isolated andidentified using suppression subtractive hybridization (SSH) and RNA-seq technology. Then,some classic regulators of skeletal muscle development, such as MSTN, IGF1and FOXO3,were picked out and their association with breast muscle traits of Pekin duck were performed.In addition, MUSTN1gene, a gene found later and may be a regulator of skeletaldevelopment, was picked out to study its molecular characteristics, to detect itsspatiotemporal expression patterns in different tissues and developmental stages and toexplore its association with muscle development. pcDNA3.1（+）-MUSTN1, the Eukaryoticexpression veyctor of MUSTN1gene, was built and was vivo transfected into the breastmuscle of Pekin duck to find out the role of MUSTN1gene in Pekin duck breast muscle. Theresults of this paper were listed as follow.1, The isolation and identification of the differentially expressed genes indifferentdevelopmental stages of Pekin duck breast muscle using suppression subtractivehybridization (SSH)The SSH libraries of breast muscle of Pekin duck at2and6weeks of age were built usingSSH technology. Totally,257differentially expressed genes, including165genes were theidentified genes in other animals and92unkown genes, was found in these two libraries. Anumber of differentially expressed genes were expressed higher in the breast muscle at6 weeks of age than that at2weeks of age and they were involved in energy metabolism andprotein synthesis. The expression levels of23differentially expressed genes in the breastmuscles of2,4,6, and8weeks of age were detected using qRT-PCR technology. Theexpression patterns of these23genes in the breast muscles of2,4,6, and8weeks of age canbe divided into3groups: those whose expression levels descended continuously, those whoseexpression levels increased firstly and then descended, and those whose expression levelsascended continuously.2, The isolation and identification of the differentially expressed genes in the breastmuscle of Pekin duck using RNA-seqAfter RNA-seq sequencing of the breast muscles and skin fats at2,4, and6weeks of ageand the quality control of initial data, the retained high quality reads of the RNA-seq datafrom the breast muscles and skin fat at2,4, and6weeks of age of Pekin duck ranged from29million to45million after sequencing and quality control of sequences and total high qualityreads (combine the6tissues) reached to218million. The bioinformatics analysis showed thatthe obtained high quality reads were distributed along with the reference genes of duck, thehigh quality reads covered89.4%of duck annotated genes and the RPKM values of thesehigh quality reads were consistent with the expression levels of genes determined byqRT-PCR. The differentially expressed genes found between tissues or developmental stagesranged from few hundred to few thousand. However, there were more the differentiallyexpressed genes between tissues than that between developmental stages. The GO and KEGGanalysis showed that differentially expressed genes were significantly enriched in differentGO terms and a number of differentially expressed genes, such as IGF1R, IGF1, MSTN,TGFβ3, TGFβ1, TGIF1, FOXO6and FOXO3were significantly enriched in those pathwaysinvolved in the regulation of skeletal development or fat deposition. For example, there were214differentially expressed genes were enriched in MAPK pathway.3, Characterization of myostatin gene (MSTN) of Pekin duck and the association of itspolymorphism with breast muscle traitsThe expression levels of MSTN gene in the breast muscle of Pekin duck at1day,2,4,6,8week of age increased from1day to4week of age. However, the expression level of thisgene at6week of age was significantly lower than that at4and6week of age using qRT-PCRtechnology. After molecular cloning and bioinformatics analysis, it was found that thefull-long CDS of Pekin duck MSTN gene contained1128bp and the protein encoded by thisgene shared high similarity with other invertebrates. MSTN gene encoded two alter splicingisoforms: the first one encoded an AA sequence with375AA, another one only contained315AA. In addition,3SNP mutation sites were found in MSTN gene CDS. The breast breadth of ducks with BB genotype was significantly higher than the ducks with AA genotype at T129CSNP site. At the T952C SNP site, ducks with the AB genotype owned significantly longerlength of fossilia ossis mastoid than those with the BB genotype.4, The expression profiles of IGF1and FOXO3and developmental differences of breastand leg muscle in Pekin duck during postnatal stages.The paraffin of breast and leg muscle at2day,2,4, and6weeks of age showed that thegrowth rate of breast muscle is lagged behind that of leg muscle. For breast muscle, the fastestgrowth stage was4~6week of age, but the fastest growth stage was2~4week of age for legmuscle. The results of using qRT-PCR detection showed that the highest expression levels ofIGF1in breast muscle and leg muscle were appeared at6weeks of age and2week of agerespectively. Conversely, the minimum values of FOXO3expression levels were appeared at6weeks of age and2week of age respectively.5, Characterization of MUSTN1gene and its relationship with skeletal muscledevelopment at postnatal stages in Pekin ducksAfter the clone of cDNA and DNA, sequence assembly and analysis of MUSTN1gene, itwas found that the full-long CDS of Pekin duck MUSTN1gene encoded a protein with78AA. MUSTN1gene entire DNA sequence was composed by3exons and2introns and anumber of transcription factor-binding sites were found in its promoter region. The expressionmaximum points of MUSTN1in breast and leg muscle were at5and3week of age. Aftervivo transfecting pcDNA3.1（+）-MUSTN1, the Eukaryotic expression vector of MUSTN1gene, into the breast muscle of Pekin duck, it can be expressed effectively in the breast muscleof Pekin duck and lead to significantly change in the expression levels of other regulators ofskeletal muscle hypertrophy.In conclusion, the differentially expressed genes in breast muscle of Pekin duck wereisolated and identified. The gene characteristics of classic regulators of skeletal muscledevelopment and the newly found MUSTN1gene were explored and their association withskeletal muscle development were detected. The results of this paper will provide a basis forexploring the mechanism of skeletal muscle development.更多还原