ube3amyomaker基因特征及其在肌纤维发育中的功能初探

Preliminary study on the characterization of ube3a and myomaker genes and their functions in muscle fiber development of Cyprinus carpio

  • 摘要:
    目的 为了解析ube3amyomaker基因在福瑞鲤2号(FFRC No. 2 strain common carp,简称FR)和从江田鱼(Congjiang strain common carp,简称CJ)肌纤维发育中的功能,探究2个品种运动性差异的分子调控机理。
    方法 采用RACE技术克隆获得FR和CJ ube3amyomaker基因cDNA全长,对其编码的蛋白性质进行了分析;并运用实时荧光定量PCR分析和原位杂交技术研究这2个基因的表达规律及定位信息。
    结果 FR和CJ ube3a基因均编码857个氨基酸,均具有一个HECT结构域。FR myomaker基因编码220个氨基酸,CJ myomaker基因编码259个氨基酸,具有一个CNMP结构域。CJ肌肉中ube3amyomaker的表达量均显著高于FR(P<0.05)。原位杂交结果显示,ube3a基因定位在肌纤维的细胞核和细胞质内,myomaker基因定位在肌纤维的细胞膜。
    结论 CJ可能由于肌肉中高ube3a表达引起较高的肌纤维蛋白质降解以及myomaker基因存在CNMP结构等原因,导致肌纤维生成较少,从而运动能力较FR弱。意义本研究为鱼类运动性差异研究提供分子基础,为进一步揭示肌纤维生长发育调控机制提供参考。

     

    Abstract: Congjiang strain common carp (Cyprinus carpio, CJ), accustomed to the GIAHS in Congjiang County, Qiandongnan Miao and Dong Autonomous Region, Guizhou Province of China, is suitable for living in shallow water bodies in paddy field due to its less locomotion behavior (local nickname “dull carp”). While the FFRC No. 2 strain common carp (FR), a nationally authorized improved aquaculture variety (registration number GS-01-003-2017) for high growth rate and survival rate, has more active locomotion behavior, resulting in a high escape potential when cultured in paddy fields. In order to explore the functions of ube3a and myomaker genes in muscle fiber development and motility differences between FR and CJ, the RACE technology was applied to clone the full-length cDNA of ube3a and myomaker genes. The properties of their encoding proteins were analyzed. The expression patterns of the two genes were explored by Real-time fluorescence quantitative PCR analysis and location information of the two genes were detected by in situ hybridization. Finally, the functions of ube3a and myomaker genes involved in the regulation of muscle fiber development were discussed. The results showed that ube3a in both common carp strains encoded 857 amino acids and contained a HECT domain. The myomaker gene in FR encoded 220 amino acids, while in CJ it encoded 259 amino acids. The CJ Myomaker protein contained a CNMP (cyclic nucleotide monophosphate-binding domain) domain. Expression analysis revealed that the transcript level of ube3a and myomaker was both significantly higher in CJ muscle compared to FR (P< 0.05). In situ hybridization results indicated that ube3a was localized in both the nucleus and cytoplasm of muscle fibers, whereas myomaker was mainly located on the cell membrane of muscle fibers. In conclusion, we speculate that the reason for the low motility ability of CJ may be that the high expression of ube3a in muscle causes the high degradation of muscle fiber protein, and the CNMP structure of myomaker gene, which may regulate the environmental adaptation, affect the fusion efficacy of myomaker function. Our findings provided a molecular basis for understanding motility differences in fish and reference for further elucidation of the regulatory mechanisms underlying muscle fiber development.

     

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