【作者】 李雪峰；

【导师】 昝林森；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2025， 硕士

【摘要】 骨骼肌的生长发育直接影响畜禽的产肉量和肉品质,因此,有关骨骼肌生成机制的研究对畜禽选育改良具有重要意义。核糖体蛋白S6(ribosomal protein S6,RPS6)基因是本课题组前期研究中发现的与肌肉发育相关的候选基因。目前尚未有研究系统探讨核糖体蛋白S6在牛骨骼肌发育中的调控作用,特别是其在成肌细胞增殖分化过程中的具体分子机制。本研究通过RNA干扰(siRNA)和重组过表达质粒技术,对牛原代成肌细胞RPS6基因进行干扰与过表达。利用Ed U、CCK-8和流式细胞术分别对DNA复制能力、细胞代谢活性以及周期分布进行检测;采用肌球蛋白重链(Myosin heavy chain,MYHC)的特异性荧光标记观察肌管融合率;同时通过RT-qPCR与Western blot检测相关标志基因的mRNA水平和蛋白水平。主要研究结果如下:1.RPS6基因在牛多种组织中广泛表达,其表达量存在显著组织差异:心中最高,脾与骨骼肌次之,肾中最低(P<0.05)。此外,RPS6的mRNA表达量在新生牛骨骼肌中显著高于成年牛(P<0.05)。在成肌细胞分化过程中(0～6天),其mRNA表达量呈现先下降后上升的趋势(P<0.05)。2.在牛成肌细胞中,RPS6干扰显著抑制细胞增殖:EdU阳性细胞比例减少(P<0.05),且G0/G1期细胞比例升高(P<0.01);而RPS6过表达则促进增殖,Ed U阳性细胞比例增加(P<0.05),并加速G0/G1期向S期转变(S期细胞比例从18.03%增至22.77%,P<0.05)。分子机制上,RPS6干扰显著下调细胞周期相关基因CCND1、MCM6和CDK1的mRNA表达(P<0.05),同时上调p21 mRNA(P<0.05)。Western blot结果显示,CDK1和CCND1蛋白表达被抑制(P<0.05),而p21蛋白表达增加(P<0.05)。相反,RPS6过表达显著增强PCNA、CDK1和CCND1的mRNA及蛋白表达(P<0.05)。3.在牛成肌细胞诱导分化过程中,RPS6干扰显著抑制分化,肌管的数量明显减少,而RPS6过表达则促进分化,细胞分化程度更高,肌管融合程度也更高。Western blot和RT-qPCR结果显示,RPS6干扰显著下调成肌分化关键基因MYOD1、MYF5、MEF2C和MYF6的mRNA表达(P<0.05),并降低MYOD1、MYF5和MEF2C蛋白水平(P<0.05)。相反,RPS6过表达显著上调了MYOD1、MYF5、MYH3和MEF2C的mRNA水平(P<0.05),并同步增强MYF5、MEF2C和MYH3的蛋白表达(P<0.05)。4.转录组测序结果显示,干扰RPS6基因后,成肌细胞分化第4天筛选到189个差异表达基因,其中上调120个,下调69个。GO功能富集分析表明,这些差异表达基因主要富集在成纤维细胞生长因子信号通路的负调控、信号转导及肌肉收缩的调节等与成肌分化密切相关的生物学过程(P<0.05)。KEGG通路分析进一步显示,差异表达基因显著富集于PPAR、NF-κB及PI3K-Akt信号通路(P<0.05)。综上所述,RPS6在心、骨骼肌等组织中广泛表达,且在新生牛骨骼肌中的表达量显著高于成年牛(P<0.05)。功能上,干扰RPS6抑制成肌细胞增殖与分化,而过表达RPS6则促进增殖并上调成肌分化标志物。RNA-seq结合分子试验(RT-qPCR、Western blot)表明,RPS6通过调控成肌分化关键基因及PPAR、PI3K-Akt和NF-κB信号通路,协同影响牛成肌细胞分化进程。上述结果表明,RPS6是调控肉牛肌肉发育的关键基因,其分子机制的阐明为肉用性状分子育种提供了新靶标。更多还原

【Abstract】 Skeletal muscle growth and development directly affects the meat yield and meat quality of livestock and poultry,therefore,research on the mechanism of skeletal muscle production is of great significance for livestock and poultry selection and improvement.The ribosomal protein S6(RPS6)gene is a candidate gene related to muscle development identified in the previous research of our group.The ribosomal protein S6(RPS6)gene is a candidate gene identified by the group based on the whole genome resequencing data of Qinchuan beef cattle,through genetic diversity analysis and selection signal analysis.No study has yet systematically explored the regulatory role of ribosomal protein S6 in bovine skeletal muscle development,especially its specific molecular mechanism in the proliferation and differentiation of adult muscle cells.In this study,we disrupted and overexpressed the RPS6 gene in bovine primary adult myoblasts by RNA interference(siRNA)and recombinant overexpression plasmid technology.The DNA replication ability,cellular metabolic activity and cycle distribution were detected by EdU,CCK-8 and flow cytometry,respectively;the myotube fusion rate was observed by specific fluorescent labelling of myosin heavy chain(MYHC);and the mRNA level and protein level of the relevant marker genes were detected by RT-qPCR and Western blot.The main findings were as follows:1.The RPS6 gene was widely expressed in a variety of bovine tissues,and there were significant tissue differences in its expression:it was highest in cardiac muscle,followed by spleen and skeletal muscle,and lowest in kidney(P<0.05).In addition,mRNA expression of RPS6 was significantly higher in skeletal muscle of neonatal cattle than in adult cattle(P<0.05).During the differentiation process of adult myoblasts(0-6 days),its mRNA expression showed a trend of decreasing and then increasing(P<0.05).2.In bovine adult myoblasts,RPS6 interference significantly inhibited cell proliferation:the proportion of EdU-positive cells decreased(P<0.05)and the proportion of cells in the G0/G1 phase increased(P<0.01),whereas RPS6 overexpression promoted proliferation,increased the proportion of Ed U-positive cells(P<0.05),and accelerated the transition from the G0/G1 phase to the S phase(the proportion of cells in the S phase increased from 18.03%to 22.77%,P<0.05).In terms of molecular mechanism,RPS6 interference significantly down-regulated the mRNA expression of cell cycle-related genes CCND1,MCM6 and CDK1(P<0.05),and up-regulated p21 mRNA(P<0.05).western blot results showed that the protein expression of CDK1 and CCND1 was suppressed(P<0.05),while the protein expression of p21 was increased(P<0.05).0.05).In contrast,RPS6 overexpression significantly enhanced mRNA and protein expression of PCNA,CDK1 and CCND1(P<0.05).3.During the induced differentiation of bovine myoblasts,RPS6 interference significantly inhibited differentiation and significantly reduced the number of myotubes,whereas RPS6 overexpression promoted differentiation,with a higher degree of cellular differentiation and myotube fusion.western blot and RT-qPCR showed that RPS6 interference significantly down-regulated the mRNA expression of the key genes for differentiation of myoblasts,MYOD1,MYF5,MEF2C and MYF6 mRNA expression(P<0.05)and reduced MYOD1,MYF5 and MEF2C protein levels(P<0.05).In contrast,RPS6 overexpression significantly up-regulated the mRNA expression of MYOD1,MYF5,MYH3,and MEF2C(P<0.05),and enhanced MYF5,MEF2C,and MYH3 protein expression(Western blot,P<0.05).4.Transcriptome sequencing results showed that 189 differentially expressed genes were detected on day 4 of myoblast differentiation after interfering with the RPS6 gene,of which120 were up-regulated and 69 were down-regulated.GO functional enrichment analysis showed that these differentially expressed genes were mainly enriched in the negative regulation of fibroblast growth factor signalling pathway,signal transduction and regulation of muscle contraction,which are closely related to myogenic differentiation.(P<0 05).KEGG pathway analysis further showed that the differentially expressed genes were significantly enriched in PPAR signalling pathway,NF-κB signalling pathway and PI3K-Akt signalling pathway(P<0.05).In summary,RPS6 was widely expressed in tissues such as skeletal muscle,cardiac muscle and liver,and the expression in skeletal muscle of neonatal cattle was significantly higher than that in adult cattle(P<0.05).Functionally,interference with RPS6 inhibited myogenic cell proliferation and differentiation,while overexpression of RPS6 promoted proliferation and up-regulated markers of myogenic differentiation.rna-seq combined with molecular assays(qPCR,western blot)showed that RPS6,by regulating key genes of myogenic differentiation and PPAR signalling pathway,PI3K-Akt signalling pathway and NF-κB signalling pathway,synergistically promoted myogenic differentiation in cattle.synergistically promoting the differentiation process of bovine myogenic cells.These results indicate that RPS6 is a key gene in the regulation of muscle development in beef cattle,and the elucidation of its molecular mechanism provides a new target for the molecular breeding of meat traits.更多还原