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

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.