家蚕细胞色素C基因的克隆及其蛋白在家蚕凋亡细胞中的释放

 【目的】细胞色素C是线粒体凋亡路径上的关键因子，通过家蚕细胞色素C基因的克隆和其蛋白在家蚕凋亡细胞中的释放研究，为家蚕细胞凋亡的研究奠定基础。【方法】利用生物信息学和分子生物学方法克隆家蚕细胞色素C基因，通过紫外线照射诱导家蚕细胞凋亡，应用流式细胞仪和Western-blotting检测家蚕凋亡细胞的线粒体膜电位变化和细胞色素C的释放。【结果】在家蚕中克隆了一个含有细胞色素C保守结构域的同源基因，该基因cDNA长570 bp，有3个外显子，开放阅读框（open reading frame，ORF）长324 bp，编码108个氨基酸，存在参与细胞凋亡时与下游凋亡蛋白酶激活因子（Apaf－1）相互作用的保守关键位点;紫外线诱导家蚕胚胎细胞BmE-SWU1凋亡后12 h，细胞线粒体跨膜电位明显下降，同时检测到了细胞色素C由线粒体向细胞质释放。【结论】在家蚕细胞凋亡中可能存在细胞色素C由线粒体释放的路径。

Cloning and Protein Release of Cytochrome C in the Apoptosis Cells of Silkworm,Bombyx mori

【Objective】 Cytochrome c plays a key role in the pathway of mitochondria apoptosis. This study on the cloning and protein release of cytochrome c in the apoptosis cells of silkworm can lay a foundation for the apoptosis research of Bombyx mori. 【Method】 Cytochrome c gene was cloned by bioinformation and molecular biology method. Apoptosis of silkworm cells was induced by UVC. The change of the mitochondrial membrane potential and the release of cytochrome c in apoptosis cells of silkworm were detected by flow cytometry and western-blotting, respectively. 【Result】 The cytochrome c gene was cloned in silkworm (Bombyx mori). The gene contains 3 exons and the length of its cDNA is 570 bp and ORF is 324 bp. The deduced protein has 108 aa and has conservative points for cytochrome c and Apaf-1 interaction related to apoptosis in silkworm. After UVC treated BmE-SWU1 cells for 12 h, the mitochondrial membrane potential decreased and cytochrome c was released from mitochondria to cytoplasm. 【Conclusion】 The cytochrome c may release from mitochondria to cytoplasm in the apoptosis cells in silkworm.