Asb14a基因在斑马鱼肌间骨发育中的作用
Role of Asb14a gene in intermuscular bone development in zebrafish(Danio rerio)
牛明慧 1王化敏 1胡瑞芹 1胡鹏 1吴智超 1陈良标1
作者信息
- 1. 上海海洋大学水产与生命学院,上海 201306;水产种质资源发掘与利用教育部重点实验室,上海 201306
- 折叠
摘要
为进一步研究肌间骨(intermuscular bone)发育的分子机制,利用CRISPR/Cas9打靶技术、茜素红染色及实时荧光定量PCR方法,研究了斑马鱼(Danio rerio)Asb14a基因敲除后肌间骨的发育情况.结果表明:本研究中成功构建了插入2 bp和缺失50 bp的Asb14a基因敲除纯合斑马鱼品系;茜素红染色显示,成年Asb14a-/-突变体与野生斑马鱼相比,肌间骨数量显著减少了 35％(P＜0.000 1),说明敲除Asb14a 基因会减少斑马鱼肌间骨的数量;实时荧光定量PCR结果显示,骨骼发育相关基因(bmp6、sp7、sox6、alpl、collala、sox6和smad1)在Asb14a-/-突变体中的表达水平发生了显著性变化,从分子角度验证了 Asb14a基因与肌间骨发育间的关联性.研究表明,Asb14a基因参与了斑马鱼肌间骨发育,敲除该基因会影响斑马鱼肌间骨的生成,这一发现为进一步研究肌间骨发育的分子机制提供了重要线索.
Abstract
In order to further probe the molecular mechanism of intermuscular bone development,the development of intermuscular bone was investigated in zebrafish(Danio rerio)in which Asb14a gene was knockouted by CRISPR/Cas9 targeting technology,alizarin red staining and real-time fluorescence quantitative PCR.The results showed that the homozygous zebrafish lines with 2 bp insertion and 50 bp deletion of Asb14a gene were successfully constructed in present study.Alizarin red staining test indicated that the number of intermuscular bones in adult Asb14a-/-mutants was found to be decreased by 35％compared with wild zebrafish(P＜0.000 1),implying that the knockout of Asb14a gene affected the formation of intermuscular bones in zebrafish.Real-time PCR analysis re-vealed that the expression levels of most bone development-related genes(bmp6,sp7,sox6,alpl,collala,sox6 and smad1)in Asb14a-/-mutants were changed significantly,verifying the association between Asb14a gene and inter-muscular bone development from a molecular perspective.The findings indicate that Asb14a gene plays an impor-tant role in the development of intermuscular bone in zebrafish.This discovery provides crucial clues for further in-vestigating the molecular mechanisms underlying the development of intermuscular bones.
关键词
斑马鱼/肌间骨/CRISPR/Cas9/Asb14a基因/纯合突变体Key words
Danio rerio/intermuscular bone/CRISPR/Cas9/Asb14a gene/homozygous mutant引用本文复制引用
基金项目
国家自然科学基金重点项目(32130109)
国家重点研发计划(2022YFD2400800)
出版年
2024
NETL
NSTL
万方数据