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大黄鱼Bmf2的基因克隆、序列分析及促细胞凋亡功能研究

Gene cloning, sequence analysis and pro-apoptotic functional study on large yellow croaker Bmf2

  • 摘要: B细胞淋巴瘤-2修饰因子(Bcl-2-modifying factor, Bmf)是Bcl-2家族中仅含BH3结构域促凋亡蛋白亚家族的一员,当凋亡刺激存在时,Bmf在真核生物细胞中启动细胞凋亡,从而在胚胎发育、器官发生、肿瘤抑制中发挥重要作用。目前,有关低等脊椎动物Bmf的研究较少,仅在斑马鱼(Danio rerio)中有相关报道。本研究通过序列比对、基因共线性分析及进化分析证实与高等脊椎动物Bmf主要以单个基因的不同剪切异构体形式存在不同,鱼类具有Bmf1Bmf2两种不同的基因。从大黄鱼(Larimichthys crocea)中克隆获得了其Bmf2LcBmf2)的开放阅读框序列,该序列全长561 bp,编码187个氨基酸。尽管LcBmf2的氨基酸序列与人和小鼠Bmf的一致性较低,但具有对Bmf功能至关重要的DLC2结合基序和BH3结构域,提示其可能具有与哺乳动物Bmf相似的功能。大黄鱼Bmf2在HEK-293T细胞系中的过表达可诱导HEK-293T细胞脱壁、形态改变以及细胞内凋亡相关Caspase 3、Caspase 8的酶活性升高,展现出较强的促凋亡功能。这是鱼类Bmf在细胞与分子水平促细胞凋亡功能的首次报道,为进一步研究Bmf在鱼类细胞凋亡中的作用及分子机制奠定了基础。

     

    Abstract: Bcl-2 modifying factor (Bmf), a member of Bcl-2 family bearing only BH3 domain, can initiate apoptosis in eukaryotic cells when apoptotic stimulation signals exist. It plays an important role in embryonic development, organogenesis and tumor suppression. Currently, Bmf from lower vertebrates has only been reported in zebrafish. Sequence alignment, gene synteny and phylogenetic analysis showed that there are two different Bmf genes, Bmf1 and Bmf2, in teleost. In this study, we cloned an open reading frame sequence of large yellow croaker Bmf2 (LcBmf2), which is 561 bp long and encoded 187 amino acids. Although the sequence identities between LcBmf2 and human or mouse Bmfs were low, LcBmf2 contains the DLC2 binding motif and conserved BH3 domain, which are highly important to Bmf function, suggesting it might exhibit similar functions to its mammalian homologue. The transient overexpression of LcBmf2 in HEK-293T cells induced cell detachment, morphology change as well as the intracellular enzyme activity increase of Caspase 3 and Caspase 8, indicating LcBmf2 prompted apoptosis in HEK-293 T cells. This is the first report on the pro-apoptotic function of a teleost Bmf at cellular and molecular level, which lays the foundation for further research on the role and molecular mechanism of Bmf in teleost cell apoptosis.

     

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