水牛波形蛋白基因克隆及其表达分析

发布时间：2018-03-17 17:20

本文选题：水牛　切入点：波形蛋白(VIM)　出处：《南方农业学报》2017年12期 　论文类型：期刊论文

【摘要】：【目的】克隆水牛波形蛋白(VIM)基因,并明确VIM基因在水牛成熟卵母细胞(MII期)和早期胚胎(2-细胞阶段)中的表达情况,为研究VIM在卵母细胞成熟及附植前胚胎发育中的作用机理打下基础。【方法】根据GenBank上已公布的水牛VIM基因mRNA序列设计引物,采用RT-PCR克隆水牛VIM基因,利用在线软件程序对克隆获得的水牛VIM基因进行生物信息学分析,同时以细胞免疫荧光试验检测VIM在水牛成熟卵母细胞(MII期)及早期胚胎(2-细胞阶段)中的表达情况。【结果】水牛VIM基因长度为1469 bp,其编码区长度1401 bp,编码466个氨基酸。水牛VIM基因序列与人类、黑猩猩、食蟹猴、家犬、马和牛的VIM基因序列具有高度的同源性,分别为93%、93%、93%、94%、94%和99%;基于VIM基因序列构建的系统发育进化树也显示水牛与牛的亲缘关系最近。水牛VIM的分子量为53.73 k D,理论等电点(p I)5.05,主要在线粒体中表达,稳定性差(不稳定系数53.65),具有较强的亲水性,无跨膜结构,不属于分泌型蛋白,其蛋白结构以α-螺旋为主。VIM在水牛成熟卵母细胞(MII期)及早期胚胎(2-细胞阶段)中均有表达,且在成熟卵母细胞中的表达量明显高于早期胚胎。【结论】水牛VIM基因编码序列具有较高的保守性,且在水牛成熟卵母细胞(MII期)中的表达量明显高于早期胚胎(2-细胞阶段)。
[Abstract]:[objective] to clone buffalo vimentin (VIM) gene, and to identify the expression of VIM gene in matured buffalo oocytes (MII) and in the early embryo (2-cell stage). In order to study the mechanism of VIM in oocyte maturation and preimplantation embryo development, a primer was designed according to the mRNA sequence of buffalo VIM gene published on GenBank, and RT-PCR was used to clone VIM gene of buffalo. The cloned buffalo VIM gene was analyzed by bioinformatics using online software program. Meanwhile, the expression of VIM in buffalo matured oocytes (MII) and in the early stage of embryo (2-cell) was detected by cellular immunofluorescence assay. [results] the length of buffalo VIM gene was 1469 BP, and the length of coding region was 1401 BP, encoding 466 ammonia. Basic acid, buffalo VIM gene sequence and human, The VIM gene sequence of chimpanzee, crab monkey, domestic dog, horse and cow is highly homologous. The phylogenetic tree based on VIM gene sequence also showed the closest relationship between buffalo and cattle. The molecular weight of buffalo VIM was 53.73 KD, and the theoretical isoelectric point was 5.05, mainly expressed in mitochondria. There was poor stability (unstable coefficient 53.65, strong hydrophilicity, no transmembrane structure and no secretory protein. The protein structure was mainly 伪 -helix in buffalo matured oocytes in MII phase) and in the early embryonic stage of 2-cell). The expression of VIM gene in matured oocytes was significantly higher than that in early embryos. [conclusion] the VIM gene coding sequence of buffalo is highly conserved, and the expression level in matured buffalo oocytes is significantly higher than that in the early embryo 2-cell stage.
【作者单位】：广西大学动物繁殖研究所/亚热带农业生物资源保护与利用国家重点实验室;
【基金】：国家自然科学基金项目(31460603) 广西研究生教育创新计划项目(YCBZ2017005)
【分类号】：S823.83

【相似文献】