斑马鱼软骨内骨化过程中骨骼的转录组测序分析  

Transcriptome analysis of bone development in zebrafish(Danio rerio)during cartilaginous endochondral ossification

作　　者：于茜 高静 纪晓 Adelino Canario 陈洁[1] YU Qian;GAO Jing;JI Xiao;Adelino Canario;CHEN Jie(International Research Center for Marine Biosciences,Ministry of Science and Technology,Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学国家海洋生物科学国际联合研究中心,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》2023年第1期95-103,共9页Journal of Dalian Ocean University

基　　金：上海海洋大学水产一流学科支持项目(A1-3201-19-3020);上海市科委“一带一路”国际联合实验室项目(19590750500)。

摘　　要：为研究斑马鱼(Danio rerio)骨骼发育时期关键调控基因及复杂的调控网络,采用阿尔辛蓝-茜素红双重染色方法对受精后50 d(day post fertilization,dpf)和65 dpf野生型斑马鱼骨骼进行染色,提取50 dpf和65 dpf斑马鱼骨骼的总RNA进行转录组测序,对差异表达基因进行筛选并进行GO功能注释和KEGG富集分析。结果表明:50 dpf斑马鱼头骨正在经历软骨内骨化,而65 dpf斑马鱼软骨内骨化过程基本完成;对50 dpf与65 dpf斑马鱼骨骼进行转录组测序,共产生41.44×109个有效数据,存在610个差异表达基因,相比50 dpf斑马鱼,65 dpf斑马鱼骨骼中软骨发育相关基因(dlx2a、foxd3和sec23b)、骨矿化调节基因(ptk2bb)、骨骼细胞分化正调控基因(dlx2a)均下调,表明其骨骼发育已较为成熟,骨细胞分化过程减少,与阿尔辛蓝-茜素红双重染色结果相一致;进一步对这些差异表达基因进行GO功能注释和KEGG富集分析,发现大部分差异表达基因与代谢网络、生殖信号通路相关;随机选取了6个差异表达基因进行实时荧光定量qRT-PCR验证,发现这些基因表达趋势与测序结果一致,这表明转录组测序结果真实可信。研究表明,鱼类与哺乳动物一样,其代谢、骨骼和生殖功能三者间存在密切联系,其内在机制为研究斑马鱼骨骼发育与疾病治疗提供了一个新的思路。In order to study the key regulated genes networks during the bone development of zebrafish(Danio rerio),whole bones of zebrafish were stained with Alcian blue and Alizarin double staining method at 50 dpf(days post fertilization)and 65 dpf to determine the developmental stages.Total RNA of bone tissues at 50 dpf and 65 dpf was extracted for transcriptome sequencing,differential expression genes(DEGs)were obtained for GO function annotation and KEGG enrichment.Double staining results showed that zebrafish was undergoing the process of endochondral ossification at 50 dpf,and basically completed this process at 65 dpf.Transcriptome results showed that 41.44×10~9 of effective data were obtained at 50 dpf and 65 dpf,with 610 DEGs.The cartilage development related genes(dlx2a,foxd3,sec23b),bone mineralization regulated gene(ptk2bb)and bone cell differentiation positive regulated gene(dlx2a)were all down regulated at 65 dpf compared to 50 dpf,indicating that the bone development was relatively matured and the process of bone cell differentiation was reduced,which was consistent with the double staining results.GO function annotation and KEGG enrichment analysis showed that most of the DEGs were enriched into the metabolic networks and reproductive signaling pathways.qPCR results with six randomly selected genes verified the reliability of the transcriptome results.Together,these results suggested that in fish,metabolism,bone development and reproduction may be closely regulated like mammals.Our results provided a new entry for the study of zebrafish bone development and disease,and a new idea for establishment of a complex disease model.

关 键 词：斑马鱼 骨骼 软骨内骨化 转录组测序 GO功能注释 KEGG富集分析 

分 类 号：S917.4[农业科学—水产科学]