Intermuscular bones (IBs) are small, hard-boned spicules located in the muscle tissue that mainly exist in the myosepta of lower teleosts, which hurt the edibleness and economic value of fish. The study of the development of IBs is very important for freshwater aquaculture fish, but the molecular mechanism of its formation and the key regulatory genes remain unclear. In this study, we first constructed two types of zebrafish mutants (the mutants losing IBs and the mutants with partial deletion of IBs) by knocking out bmp6. We then carried out a transcriptomic analysis to reveal the role of bmp6 in the developmental mechanism of IBs; we used the caudal musculoskeletal tissues of these mutants and wild-type zebrafish at three development stages (20, 45, and 60 dph) to perform transcriptomic analysis. The results showed that the deficiency of bmp6 upregulated sik1 and activated the TNF-A signaling via the NF-KB pathway, which inhibited the development of osteoblasts and promoted osteoclast formation, thereby inhibiting the formation of IBs. These results provided insights to understand the role of bmp6 in the development of IBs in zebrafish and are useful for selective breeding of IBs in cyprinids.
As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.
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