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Cloning and expression of MyoG gene from Hu sheep and identification of its myogenic specificity

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Abstract

This experiment was conducted to explore the biological functions of myogenin (MyoG) gene. MyoG gene was cloned from genome of Hu sheep by overlap extension PCR. Then, pEGFP-C1–MyoG and pcDNA3.0–MyoG fusion expression vectors was constructed and pEGFP-C1–MyoG vector had been transfected into NIH-3T3 cells by liposomes-mediated method, and MyoG was detected in vitro by RT-PCR,western blotting and its subcellular localization by EGFP marker. pcDNA3.0–MyoG was transfected into goat embryonic fibroblasts (GEF) cells in order to detect the myogenic function of MyoG in vitro. Then pEGFP-C1–MyoG plasmid was injected into the testes of sheep and goat, respectively, to produce the transgenic generation. The results showed that the length of MyoG coding region of Hu sheep was 675 bp, encoding 224 amino acids. Compared with goat, cattle, pig and rat, the sequence homology of sheep MyoG cDNA was 99.26, 97.04, 92.00, and 87.70 %, respectively. The bioinformatics prediction showed that MyoG protein contained a typical bHLH structure, but without a short signal peptide, revealing that MyoG protein might be a non-secretory protein. The result of RT-PCR and western blotting demonstrated that MyoG could be expressed successfully in the transfected cells in vitro and the MyoG protein was located in nucleus. The positive transfected GEF cells with pcDNA3.0–MyoG were found to express desmin protein. The positive rates of transgenic sheep and transgenic goat were 7.1 and 7.4 % in F1 generation, respectively. Conclusively, MyoG cDNA from Hu sheep had been cloned successfully. The subcellular localization and myogenic activity of MyoG were exactly detected on the basis of multiple biological analyses, which expanded our understanding of the biological function of MyoG.

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Acknowledgments

The study was supported by National New varieties of GMO cultivation of major projects (2011ZX08008-003; 2009ZX08008-003B) in China; A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China

    Zhentao Zhang, Feng Xu, Yani Zhang, Wei Li, Yanhui Yin, Caiye Zhu, A. K. Elsayed & Bichun Li

  2. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

    Lixin Du

  3. College of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt

    A. K. Elsayed

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  1. Zhentao Zhang
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  2. Feng Xu
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  3. Yani Zhang
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  4. Wei Li
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  5. Yanhui Yin
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  6. Caiye Zhu
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  7. Lixin Du
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  9. Bichun Li
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Correspondence to Bichun Li.

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Zhentao Zhang and Feng Xu have contributed equally to this study.

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Zhang, Z., Xu, F., Zhang, Y. et al. Cloning and expression of MyoG gene from Hu sheep and identification of its myogenic specificity. Mol Biol Rep 41, 1003–1013 (2014). https://doi.org/10.1007/s11033-013-2945-0

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  • DOI: https://doi.org/10.1007/s11033-013-2945-0

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