摘要
【目的】探究硬骨鱼类前胶原蛋白酶增强因子(PCOLCE)的2种亚型(pcolcea和pcolceb)在斑马鱼骨骼发育中的作用。【方法】采用生物信息学分析、原位杂交、荧光定量PCR、CRISPR/Cas9基因编辑及显微型计算机断层扫描等分析技术和实验手段以明确斑马鱼pcolcea和pcolceb基因的序列特征、蛋白结构、组织表达以及功能缺失对骨骼发育的影响。【结果】pcolcea和pcolceb基因主要在脊椎、头部和胸鳍表达。与野生型斑马鱼相比,pcolcea^(-/-)斑马鱼和pcolcea^(-/-);pcolceb^(-/-)双突变斑马鱼脊椎骨的密度显著降低,而pcolceb^(-/-)斑马鱼与野生型相比无显著性差异。在pcolcea^(-/-)突变体的尾部椎骨中,bmp1b、col1a1a、col1a2、runx2b、alpl、sp7基因的表达显著下调,在pcolcea^(-/-);pcolceb^(-/-)双突变体的尾部椎骨中,bmp1b、col1a1a、col1a1b、col1a2、runx2b、entpd5a、bglap、sp7基因的表达均显著低于野生型斑马鱼。【结论】pcolcea基因功能的丧失会影响斑马鱼骨骼组织的正常矿化,且pcolcea和pcolceb基因的同时缺失会显著加重该表型。本研究揭示了pcolcea和pcolceb在斑马鱼骨骼发育中的作用,为进一步研究鱼类骨骼发育的分子调控机制提供了科学依据。
Procollagen C-proteinase enhancer protein(PCOLCE)is a secreted protein that enhances the activity of procollagen C-proteinase,thereby facilitating the maturation of collagen and influencing the process of bone formation.Down-regulation of pcolce expression compromises the maturation of collagen,ultimately leading to a reduction in bone mass.In teleost fish,the occurrence of genomic duplication events has led to the emergence of two paralogs of pcolce,designated pcolcea and pcolceb,whose functional roles in skeletal development remain incompletely characterized.To delineate the functional divergence of pcolcea and pcolceb in zebrafish(Danio rerio)osteogenesis,this study employed a multi-faceted approach integrating computational analysis of sequence features,spatiotemporal expression profiling,and phenotypic characterization of targeted mutants.The results indicated that pcolcea and pcolceb comprise 8 and 9 exons,respectively,with pcolceb being significantly longer;both genes were predominantly expressed in the vertebrae,head,and pectoral fins.Through targeted gene knockout,we generated pcolcea^(-/-)and pcolceb^(-/-)homozygous mutants.The pcolcea^(-/-)mutant carried a 229-bp deletion in exon 1,while the pcolceb^(-/-)mutant carried a 7-bp deletion in exon 3.Compared to wild-type D.rerio,pcolcea^(-/-)and pcolcea^(-/-);pcolceb^(-/-)double mutant D.rerio exhibited a marked reduction in vertebral tissue mineral density,whereas pcolceb^(-/-)D.rerio showed no significant difference.Subsequently,the expression of genes related to bone development(runx2b,entpd5a,alpl,bglap,sp7),collagen development(col1a1a,col1a1b,col1a2),and downstream effector genes(bmp1a,bmp1b)was quantified by RT-qPCR in the caudal vertebrae of the mutants.RT-qPCR results showed that expression of bmp1b,col1a1a,col1a2,runx2b,alpl,and sp7 was significantly downregulated in pcolcea^(-/-)mutants.In pcolcea^(-/-);pcolceb^(-/-)double mutants,the expression of bmp1b,col1a1a,col1a1b,col1a2,runx2b,entpd5a,bglap,and sp7 was significantly lower than in wild-type D.rerio.Collectively,these results demonstrated that loss of pcolcea impaired skeletal tissue mineralization in D.rerio,and this phenotype was significantly exacerbated by the concurrent deletion of pcolceb.This study elucidates the distinct roles of pcolcea and pcolceb in D.rerio bone development and provides a foundation for understanding the molecular mechanisms governing skeletal formation in fish.
作者
王文秀
陈宇龙
肖正雨
高泽霞
聂春红
WANG Wenxiu;CHEN Yulong;XIAO Zhengyu;GAO Zexia;NIE Chunhong(Key Lab of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affairs,Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt,Ministry of Education,College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Hubei Hongshan Laboratory,Wuhan 430070,China;Hubei Province Famous Fish Breeding and Healthy Aquaculture Engineering Technology Research Center,Wuhan 430070,China)
出处
《水产学报》
北大核心
2026年第1期22-33,共12页
Journal of Fisheries of China
基金
国家自然科学基金青年科学基金(32202917)
国家自然科学基金重点项目(32330108)
中央高校基本科研业务费专项(2662023SCQD002)
湖北省自然科学基金(2022CFB819)。
