水牛热休克蛋白70基因克隆及其表达分析

Cloning and expression analysis of buffalo heat shock protein 70 gene

  • 摘要: 【目的】克隆水牛热休克蛋白70基因(HSP70)并进行生物信息学分析,同时明确HSP70基因在水牛成熟卵母细胞(MII期)和2-细胞期胚胎中的表达情况,为研究HSP70在水牛卵母细胞成熟及着床前早期胚胎发育中的作用机理提供理论依据。【方法】根据NCBI已公布的水牛HSP70基因mRNA预测序列(MH814759.1)设计引物,通过RTPCR克隆水牛HSP70基因,运用DNAStar、MEGA 7.0、TMHMM 2.0及SignalP 4.1 Server等在线软件进行生物信息学分析;同时采用细胞免疫荧光分析HSP70在水牛成熟卵母细胞及2-细胞期胚胎中的表达情况。【结果】水牛HSP70基因编码区(CDS)长度为1926 bp,共编码641个氨基酸残基。克隆获得的水牛HSP70基因序列与NCBI已公布水牛HSP70基因mRNA预测序列(MH814759.1)的相似性高达97.7%;与牛、家犬、山羊、小鼠、绵羊和大鼠的HSP70基因序列具有较高的相似性,其中与牛的相似性高达99.1%。基于HSP70基因核苷酸序列相似性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HSP70分子量为70.27 kD,分子式为C3088H4967N8670972S15,理论等电点(pI)为5.67,不稳定系数为32.84,属于稳定蛋白;不存在跨膜结构及信号肽,主要定位于细胞质(60.9%)、细胞核(30.4%)及线粒体(8.7%),其亲水性较强;水牛HSP70蛋白结构以α-螺旋和β-折叠为主。HSP70在水牛成熟卵母细胞及2-细胞期胚胎中均有表达,且主要集中在细胞质。【结论】水牛HSP70基因在进化过程中具有高度的保守性,在水牛成熟卵母细胞和着床前早期胚胎中均有表达,且主要集中在细胞质,属于结构型HSP70。

     

    Abstract: 【Objective】 The heat shock protein 70 gene of buffalo (HSP70)was cloned and analyzed by bioinformatics. Meanwhile,its expression was analyzed in buffalo mature oocytes(MII stage)and 2-cell stage embryos,which provided a theoretical basis for studying the mechanism of HSP70 in the maturation of oocytes and pre-implantation embryos development.【Method】 Primer was designed according to the published mRNA sequence of buffalo gene HSP70 (MH814759.1) in GenBank. Bioinformatics analysis was performed using online software such as DNAStar,MEGA 7.0,TMHMM 2.0, and SignalP 4.1 Server. Meanwhile,the immunofluorescence assay was used to detect the expression of HSP70 in mature oocytes and 2-cell stage embryos.【Result】 The coding region(CDS)of buffalo gene HSP70 was 1926 bp, and 641 amino acids were encoded. The cloned buffalo gene HSP70 had 97.7% homology with the published buffalo HSP70 mRNA prediction sequence(MH814759.1). And it had high homology with that of Bos taurus,Canis lupus familiaris,Capra hircus,Mus musculus,Ovis ariesis,Rattus norvegicus,among which the homology of B. taurus was as high as 99.1%. The phylogenetic tree based on the similarity of the nucleotide sequence of HSP70 also showed that the relationship between the buffalo and the B. taurus was the closest. The molecular weight of buffalo HSP70 was 70.27 kD,molecular formula was C3088H4967N8670972S15, the theoretical isoelectirc point(pI)was 5.67,which showed the HSP70 belonged to stabilized protein(instability index was 32.84). There was no transmembrane structure and signal peptide,mainly located in the cytoplasm(60.9%),nucleus(30.4%)and mitochondria(8.7%),and its hydrophilicity was strong. The structure of buffalo HSP70 protein was mainly α-helix and β-sheet. HSP70 was expressed in buffalo MII oocytes and 2-cell stage embryos,and was mainly concentrated in the cytoplasm.【Conclusion】 The buffalo gene HSP70 is highly conserved during the evolution process. It is expressed in mature buffalo oocytes and early embryos before implantation,and is mainly concentrated in the cytoplasm. It belongs to structural HSP70.

     

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