草鱼BMP6基因克隆与表达特征分析

Cloning and expression characteristics analysis of BMP6 gene in grass carp (Ctenopharyngodon idella)

  • 摘要:
    目的 探究草鱼骨形态发生蛋白6 (BMP6)基因的序列结构及其表达模式。
    方法 通过RACE技术克隆鉴定草鱼BMP6基因,利用生物信息学方法分析其氨基酸序列、系统进化关系及蛋白结构域等;采用实时荧光定量PCR (RT-qPCR)检测BMP6在不同组织中的表达水平;利用RNA荧光原位杂交技术对尾部肌肉组织(含肌间骨)的BMP6的表达进行定位。
    结果 本研究获得草鱼BMP6基因cDNA全长4 942 bp,编码417个氨基酸。多序列比对及系统进化分析表明,草鱼BMP6与团头鲂亲缘关系最近,氨基酸序列一致性和相似性分别达97.6%和99.3%。蛋白结构分析显示,草鱼BMP6具有典型TGFβ_propeptide和TGFβ结构域,包含保守的RTAR蛋白裂解位点、7个半胱氨酸残基及1个N-糖基化位点。组织表达分析发现,BMP6在鳃中表达最高,肝脏、肠道、心脏次之,在肾脏、血液和性腺中表达较低。RNA荧光原位杂交结果进一步证实,BMP6在草鱼尾部肌肉组织中的肌间骨处存在特异性高表达。
    结论 草鱼BMP6具有典型的BMP家族保守特征,且在肌间骨处显著高表达,提示其可能在肌间骨的稳态维持过程中发挥重要作用。本研究为进一步探索草鱼肌间骨发育的分子机制及创制少(无)肌间骨草鱼新种质提供重要的分子基础。

     

    Abstract: Bone morphogenetic protein 6 (BMP6) plays a critical role in the skeletal development of vertebrates, yet its function in the formation of intermuscular bones (IBs) in fish remains unclear. In Ctenopharyngodon idella, the presence of numerous IBs diminishes fillet quality and economic value, which underscores the necessity of molecular research to facilitate genetic improvement. To investigate the structural features and expression characteristics of BMP6 gene in C. idella, the full-length cDNA sequence of BMP6 was cloned using RACE, followed by bioinformatics analysis of its amino acid sequence, phylogenetic relationships, and protein domains. Tissue-specific expression profiles were detected by RT-qPCR, and the spatial localization of BMP6 transcripts in tail muscle tissue (including intermuscular bones) was determined by RNA fluorescence in situ hybridization. The full-length cDNA sequence of C. idella BMP6 was 4 942 bp, encoding 417 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that C. idella BMP6 is most closely related to that of Megalobrama amblycephala, with 97.6% identity and 99.3% similarity at the amino acid level. Protein structure analysis indicated that C. idella BMP6 contains conserved TGFβ_propeptide and TGFβ domains, along with a conserved proteolytic cleavage site (RTAR), seven cysteine residues, and one N-glycosylation site. Tissue expression analysis showed that BMP6 was most highly expressed in the gill, followed by the liver, intestine, and heart, while lower expression levels were observed in the kidney, blood, and gonad. RNA fluorescence in situ hybridization further confirmed significantly high expression of BMP6 in intermuscular bones of the tail muscle tissue. This study provides important insights into the molecular mechanisms underlying intermuscular bone development in C. idella and establishes a theoretical foundation for the development of a new strain lacking intermuscular bones in C. idella.

     

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