【摘要】 【目的】研究胚胎期北京鸭腿肌的发育规律并进一步筛选关键调控基因。【方法】选取第11(E11)、17(E17)和23(E23)胚龄的北京鸭胎胚，测定胚胎重和腿肌重，并制备腿肌组织HE染色切片用于组织形态学观察。取不同时期北京鸭腿肌组织进行转录组测序，通过DESeq2 R软件包对转录组数据进行差异表达基因（DEGs）筛选，进一步对差异表达基因进行KEGG通路富集分析及Mfuzz时序聚类分析；以腿肌率为表型，对不同时期转录组数据开展加权基因共表达网络分析。随机选取5个DEGs进行实时荧光定量PCR验证。【结果】北京鸭胚胎重、腿肌重和腿肌率随胚龄的增长而增加，且不同胚龄间差异显著（P<0.05）。组织形态学观察发现，E11时期北京鸭腿肌中存在大量单个肌细胞，E17时期可观察到肌束结构，至E23时期可见成熟肌纤维。E11 vs E17、E17 vs E23、E11 vs E23组中差异表达基因的数量分别为2 030、3 487和5 406个。3组中筛选到的差异表达基因均显著富集到肌肉细胞中的细胞骨架通路和过氧化物酶体增殖物激活受体信号通路等9条KEGG代谢通路。通过加权基因共表达网络分析筛选到与腿肌率呈显著正相关的yellow模块，结合Mfuzz时序聚类分析得到3个表达趋势与腿肌率一致的基因簇。共有9个基因在上述yellow模块和3个基因簇中均出现。其中，转谷氨酰胺酶2(transglutaminase 2,TGM2)、连接蛋白2(junctophilin 2, JPH2)、干扰素调节因子2结合蛋白2(interferon regulatory factor 2 binding protein 2, IRF2BP2)、线粒体核糖体蛋白L55(mitochondrial ribosomal protein L55, MRPL55)和夜蛋白（nyctalopin, NYX）5个基因的表达水平与腿肌率呈高度正相关（r>0.7）。实时荧光定量PCR结果与转录组测序一致。【结论】本研究分析了胚胎期北京鸭腿肌的发育规律，并鉴定到5个潜在的调控基因TGM2、JPH2、IRF2BP2、MRPL55和NYX。本研究结果为阐明北京鸭腿肌发育的分子机制提供了科学依据。更多还原

【Abstract】 【Objective】 This study aimed to investigate the developmental pattern of leg muscle of Pekin ducks in embryonic stage and further screen key regulatory genes involved in its development.【Method】 In this study, Pekin duck embryos at embryonic days 11(E11), 17(E17), and 23(E23) were selected. Embryo weight and leg muscle weight were measured, and HE stained sections of leg muscle were prepared for histomorphological observation. RNA-Seq was performed on the leg muscle of Pekin ducks at different stages. Differentially expressed genes(DEGs) were screened from the transcriptome data using the DESeq2 R package. Furthermore, KEGG enrichment analysis and Mfuzz temporal clustering analysis were conducted on the DEGs. In addition, weighted gene coexpression network analysis was conducted on the transcriptome data of the different stages using leg muscle rate as the phenotype. 5 DEGs were randomly selected for Real-time quantitative PCR verificaton. 【Result】 The embryo weight, leg muscle weight,and leg muscle ratio of Pekin ducks increased with the growth of embryonic age, and there were significant differences among different embryonic ages(P<0.05). Histological observation revealed that during the E11 stage, there were a large number of individual myocytes in the leg muscles of Pekin ducks. By the E17 stage, the muscle bundle structure could be observed, and by the E23 stage, mature muscle fibers could be seen. DEGs analysis indicated that the numbers of DEGs in E11 vs E17, E17 vs E23, and E11 vs E23 groups were 2 030, 3 487, and 5 406, respectively. The DEGs identified in three groups were significantly enriched in 9 KEGG pathways, including the cytoskeleton pathway in muscle cells and the peroxisome proliferator-activated receptor signaling pathway. Subsequently, a yellow module significantly and positively correlated with leg muscle rate was identified via weighted gene coexpression network analysis. Combined with Mfuzz temporal clustering analysis, 3 gene clusters with expression trends consistent with changes in leg muscle rate were obtained. A total of 9 genes were present in both the yellow module and 3 gene clusters. Among these, the expression of five genes exhibited a strong positive correlation with leg muscle rate(r>0.7),including transglutaminase 2(TGM2),junctophilin 2(JPH2),interferon regulatory factor 2 binding protein 2(IRF2 BP2),mitochondrial ribosomal protein L55(MRPL55),and nyctalopin(NYX). The results of Real-time quantitative PCR were consistent with thoes of RNA-Seq. 【Conclusion】 This study analyzed the developmental pattern of leg muscles of Pekin ducks during the embryonic stage and identified 5 potential regulatory genes: TGM2,JPH2,IRF2 BP2,MRPL55,and NYX. The results provided a scientific basis for elucidating the molecular mechanisms underlying leg muscle development in Pekin ducks.更多还原