Cloning and expression analysis of buffalo heat shock protein 70 gene

【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 C₃₀₈₈H₄₉₆₇N_8670972S₁₅, 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.