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Fish Physiology and Biochemistry

, Volume 43, Issue 2, pp 435–454 | Cite as

Cloning, expression profiling and promoter functional analysis of Bone morphogenetic protein 6 and 7 in tongue sole (Cynoglossus semilaevis)

  • Qian Ma
  • Wenrong Feng
  • Zhimeng Zhuang
  • Shufang Liu
Article

Abstract

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836 bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1–4 days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5′-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of −272 to +28 and −740 to −396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

Keywords

Cloning Gene expression pattern Promoter transcriptional activity Bone morphogenetic protein Cynoglossus semilaevis Early developmental stages 

Notes

Acknowledgments

The authors are very grateful to Haiyang hatchery station (Qingdao, Shandong Province) for their support in fish culture and sampling. The research was financially supported by the National Natural Science Foundation of China (Grant No.: 31201981), Special Scientific Research Funds for Central Non-profit Institutes, Chinese Academy of Fishery Sciences (2016RC-LX02) and the Applied Basic Research Programs of Qingdao (14-2-4-15-jch).

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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Qian Ma
    • 1
    • 2
  • Wenrong Feng
    • 1
  • Zhimeng Zhuang
    • 1
    • 3
  • Shufang Liu
    • 1
    • 2
  1. 1.Yellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdaoChina
  2. 2.Function Laboratory for Marine Fisheries Science and Food Production ProcessesQingdao National Laboratory for Marine Science and TechnologyQingdaoChina
  3. 3.Function Laboratory for Marine Biology and BiotechnologyQingdao National Laboratory for Marine Science and TechnologyQingdaoChina

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