摘要
为了克隆努比亚山羊(Capra nubiana)同源异形盒转录因子1(prospero-related homeobox protein 1,PROX1)基因和检测过表达PROX1基因对努比亚山羊骨骼肌成肌细胞分化和肌纤维类型转化相关基因mRNA表达的影响,为研究PROX1基因在骨骼肌发育中的调控作用提供参考,本研究以努比亚山羊背最长肌组织cDNA为模板克隆PROX1基因,构建pEGFP-N1-PROX1真核表达载体,转染至努比亚山羊骨骼肌成肌细胞,24 h后更换分化培养基,收集分化6 d的细胞,通过RT-qPCR检测过表达PROX1基因后细胞分化标志基因、NOTCH信号通路相关基因以及骨骼肌肌纤维类型相关基因的表达情况。研究结果表明努比亚山羊PROX1基因编码区(coding sequence,CDS)序列长度为2214 bp,编码含737个氨基酸的多肽。RT-qPCR结果表明PROX1基因可以通过抑制NOTCH信号通路,上调细胞分化标志基因表达水平进而促进成肌细胞的分化(P<0.05),并介导CaN/NFAT信号通路,显著下调MYH2和MYH4基因的mRNA表达水平(P<0.05)。本研究初步确定努比亚山羊PROX1基因参与调控骨骼肌成肌细胞的分化和肌纤维类型的转化,研究结果为进一步丰富肌肉生长发育的分子机制提供理论数据。
The aim of this study was to clone the prospero-related homeobox protein 1(PROX1)gene of the Nubian goat(Capra nubiana),and to detect the mRNA expression of genes related to myoblast differentiation and the transformation of myofiber types after overexpressing PROX1 gene in myoblasts of Nubian goat.This study aimed to provide reference for studying the regulatory role of the PROX1 gene in ske-letal muscle development.The cDNA of the longest dorsal muscle tissue of the Nubian goat was used as the template to clone the PROX1 gene,and a eukaryotic expression vector pEGFP-N1-PROX1 was successfully constructed and identified.These constructs were then transfected into Nubian goat myoblasts.The differentiation medium was changed 24 hours later,and the cells were collected after differentiation for 6 days.The expression of marker genes for cellular differentiation,genes related to the NOTCH signaling pathway,and genes related to the types of myofibers in skeletal muscles were examined by RT-qPCR in the cells.The results of the study showed that the coding sequence(CDS)length of the PROX1 gene was 2214 bp in the Nubian goat,encoding a polypeptide with 737 amino acids.The results of the RT-qPCR showed that the PROX1 gene promoted myoblast differentiation by inhibiting the NOTCH signaling pathway and up-regulating the expression level of cell differentiation marker genes(P<0.05).The PROX1 gene also mediated the CaN/NFAT signaling pathway and significantly down-regulated the mRNA expression level of MYH2 and MYH4 genes(P<0.05).This study preliminarily determines that the PROX1 gene regulates myoblast differentiation and the transformation of muscle fiber types.The research results provide theoretical data for further enriching the understanding of the molecular mechanisms of muscle growth and development.
作者
刘雨帆
邹菊红
何海恩
张叁保
黄艳娜
蒋钦杨
LIU Yufan;ZOU Juhong;HE Hai′en;ZHANG Sanbao;HUANG Yanna;JIANG Qinyang(College of Animal Science and Technology,Guangxi University,Nanning,530004)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2024年第7期1214-1224,共11页
Genomics and Applied Biology
基金
国家现代农业产业技术体系广西创新团队建设项目(nycytxgxcxtd-2021-09)资助。
