猪SELW基因对骨骼肌卫星细胞分化的影响
Effect of Pig(Sus scrofa)SELW Gene on the Differentiation of Skeletal Muscle Satellite Cells
胡荣斌 1吴兴凤 1潘志洪 1李莉 1何逸懿 1徐娥1
作者信息
- 1. 贵州大学动物科学学院动物营养与饲料研究所/高原山地动物遗传育种与繁殖教育部重点实验室,贵阳 550025
- 折叠
摘要
硒蛋白W(selenoprotein W,SELW)是骨骼肌中重要的硒蛋白之一,对细胞分化等多种生物学功能具有重要作用,为探究SELW基因对猪(Sus scrofa)骨骼肌卫星细胞分化的影响.选取3头7日龄健康的大白猪,采集心、肝、脾、肺、肾、背肌、腿肌7个组织样提取RNA,qPCR检测SELW基因在不同组织中的相对表达量.运用在线软件对猪SELW蛋白质进行生物信息学分析.克隆猪SELW基因(GenBank No.NM_213977)CDS全长序列,构建过表达SELW基因重组慢病毒载体并感染骨骼肌卫星细胞,嘌呤霉素筛选获得SELW稳定表达细胞系,通过亚细胞定位确定SELW蛋白的表达部位,qPCR和Western blot检测SELW基因及蛋白的表达水平.收集诱导分化0和48h的细胞样,qPCR检测配对盒子3基因(paired box 3,PAX3)、PAX7、肌源性因子5基因(myogenic factor 5,MYF5)、生肌决定因子基因(myogenic determinant,MYOD)、肌细胞生成素基因(myocyte generating factor,MYOG)的表达量.结果表明,SELW基因在猪7个不同组织中均有表达,在心脏和背肌中表达量极显著高于其他组织(P＜0.01).SELW蛋白分子式为C421H677N109O119S3Se1,相对分子质量为9 344.81 D,理论等电点为9.15,属于稳定蛋白质.遗传进化分析可知猪SELW基因与人(Homo sapiens)、恒河猴(Macaca mulatta)、苏门答腊猩猩(Pongo abelii)相似性最高.亚细胞定位结果表明,SELW蛋白定位于细胞核和细胞质.qPCR和Western blot检测SELW过表达效率,SELW-OE组表达量极显著高于SELW-NC组(P＜0.01),表明SELW基因稳定表达细胞系构建成功.qPCR结果表明,诱导分化0h时SELW-OE组的PAX7和MYF5基因的表达量极显著上调,而MYOD基因表达量显著下调(P＜0.05),MYOG基因的表达量极显著下调(P＜0.01);诱导分化48h后PAX3基因的表达量显著上调(P＜0.05),PAX7、MYF5、MYOD和MYOG基因的表达极显著上调(P＜0.01),表明SELW基因过表达后可促进与骨骼肌细胞分化相关基因的表达.本研究为进一步探讨SELW基因对猪肌肉生长发育的分子调控机制提供基础资料.
Abstract
Selenoprotein W(SELW)is one of the important selenoproteins in skeletal muscle,which has important roles in cell differentiation and other biological functions.To investigate the effect of SELW gene on the differentiation of skeletal muscle satellite cells in pigs(Sus scrofa),three 7-day-old healthy white pigs were selected,and 7 tissue samples of heart,liver,spleen,lung,kidney,dorsal muscle and leg muscle were collected to extract RNA.qPCR was performed to detect the relative expression of SELW gene in different tissues.Bioinformatics analysis of porcine SELW proteins was performed using online software.The full-length sequence of the CDS region of porcine SELW gene(GenBank No.NM_213977)was cloned,a recombinant lentiviral vector overexpressing SELW gene was constructed and infected with skeletal muscle satellite cells,SELW stably expressed cell lines were obtained by puromycin screening,SELW protein expression sites was determined by subcellular localization,and SELW gene and its protein expression levels were detected by qPCR and Western blot.Cell samples were collected at 0 and 48 h of induction differentiation,and qPCR was performed to detect the expression of paired box 3(PAX3),PAX7,myogenic factor 5(MYF5),myogenic determinant(MYOD),and myocyte generating factor(MYOG)genes.The results showed that SELW genes were expressed in 7 different tissues of pigs,and the expression was extremely significant differenct in heart and dorsal muscle than in other tissues(P＜0.01).The molecular formula of SELW protein was C421H677N109O119S3Se1,the relative molecular weight was 9 344.81 D,and the theoretical isoelectric point was 9.15,which was a stable protein.Genetic evolutionary analysis showed that pig SELW gene had the highest similarity with human(Homo sapiens),rhesus monkey(Macaca mulatta)and Sumatran orangutan(Pongo abelii).Subcellular localization results indicated that SELW proteins were localized in the nucleus and cytoplasm of cells.qPCR and Western blot results showed that the expression of SELW-OE group was very significant higher than that of SELW-NC group(P＜0.01),indicating the successful construction of SELW gene stably expressing cell lines.qPCR results showed that the expression of PAX7 and MYF5 genes in SELW-OE group were extremely significantly upregulated at 0 h of induction differentiation,while MYOD gene expression was significantly down-regulated(P＜0.05),and MYOG gene expression was extremely significantly down-regulated(P＜0.01).The expression of PAX3 gene was significantly upregulated(P＜0.05)and the expression of PAX7,MYF5,MYOD and MYOG genes were extremely significantly upregulated(P＜0.01)after 48 h of induction of differentiation.It was shown that overexpression of SELW gene could promote the expression of genes related to skeletal muscle cell differentiation.It provides basic information for further study of the molecular regulation mechanism of SELW gene on muscle growth and development in pigs.
关键词
硒蛋白W(SELW)/重组慢病毒载体/猪/骨骼肌卫星细胞Key words
Selenoprotein W(SELW)/Recombinant lentiviral vector/Pig/Skeletal muscle satellite cells引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据