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)

作　　者：牛明慧 王化敏 胡瑞芹 胡鹏[1,2] 吴智超[1,2] 陈良标 NIU Minghui;WANG Huamin;HU Ruiqin;HU Peng;WU Zhichao;CHEN Liangbiao(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Aquatic Germplasm Resources Exploration and Utilization,Ministry of Education,Shanghai 201306,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]水产种质资源发掘与利用教育部重点实验室,上海201306

出　　处：《大连海洋大学学报》2024年第2期259-265,共7页Journal of Dalian Ocean University

基　　金：国家自然科学基金重点项目(32130109);国家重点研发计划项目(2022YFD2400800)。

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<0.0001),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。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<0.0001),implying that the knockout of Asb14a gene affected the formation of intermuscular bones in zebrafish.Real-time PCR analysis revealed 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 intermuscular bone development from a molecular perspective.The findings indicate that Asb14a gene plays an important role in the development of intermuscular bone in zebrafish.This discovery provides crucial clues for further investigating 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 

分 类 号：S917.4[农业科学—水产科学]