联系我们 加入收藏
 
年期检索 高级检索
33
2025年8月3日 星期日
农业生物技术学报  2024, Vol. 32 Issue (1): 168-179    DOI: 10.3969/j.issn.1674-7968.2024.01.015
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
SELW基因对骨骼肌卫星细胞分化的影响
胡荣斌, 吴兴凤, 潘志洪, 李莉, 何逸懿, 徐娥*
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
Effect of Pig (Sus scrofa) SELW Gene on the Differentiation of Skeletal Muscle Satellite Cells
HU Rong-Bin, WU Xing-Feng, PAN Zhi-Hong, LI Li, HE Yi-Yi, XU E*
Key Laboratory of Genetic Breeding and Reproduction of Plateau Mountain Animals, Ministry of Education/Institute of Animal Nutrition and Feed, School of Animal Science, Guizhou University, Guiyang 550025, China
全文: PDF (11278 KB)   HTML (1 KB) 
输出: BibTeX | EndNote (RIS)      
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48 h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0 h时SELW-OE组的PAX7MYF5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48 h后PAX3基因的表达量显著上调(P<0.05),PAX7MYF5MYODMYOG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。
服务
把本文推荐给朋友
加入我的书架
加入引用管理器
E-mail Alert
RSS
作者相关文章
胡荣斌
吴兴凤
潘志洪
李莉
何逸懿
徐娥
关键词 硒蛋白W (SELW)重组慢病毒载体骨骼肌卫星细胞    
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.
Key wordsSelenoprotein W (SELW)    Recombinant lentiviral vector    Pig    Skeletal muscle satellite cells
收稿日期: 2022-10-10     
ZTFLH:  S828.8  
基金资助:国家自然科学基金(31860648)
通讯作者: *exu@gzu.edu.cn   
引用本文:   
胡荣斌, 吴兴凤, 潘志洪, 李莉, 何逸懿, 徐娥. 猪SELW基因对骨骼肌卫星细胞分化的影响[J]. 农业生物技术学报, 2024, 32(1): 168-179.
HU Rong-Bin, WU Xing-Feng, PAN Zhi-Hong, LI Li, HE Yi-Yi, XU E. Effect of Pig (Sus scrofa) SELW Gene on the Differentiation of Skeletal Muscle Satellite Cells. 农业生物技术学报, 2024, 32(1): 168-179.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.01.015     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I1/168
 
版权所有 © 2014 《农业生物技术学报》编辑部   京ICP备11035905号-3
地址:北京市海淀区圆明园西路2号中国农业大学生命科学楼1053室 邮编:100193
电话:010-62733684 传真:010-62731615 E-mail: nsjxb@cau.edu.cn