水牛SFRP1基因序列分析、真核表达载体构建及组织表达分析OA北大核心CSTPCD

[Objective]The aim of this study was to obtain the CDS region sequence of the secreted fizzled-related protein 1(SFRP1)gene in buffalo,predict the structure and function of the encoded protein,construct the eukaryotic expression vector of SFRP1 gene,and detect the expression of SFRP1 gene in different tissues of buffalo,laying a foundation for exploring the role of SFRP1 gene in the growth and development of buffalo.[Method]Using the cDNA of ovarian tissue as template,the CDS region sequence of SFRP1 gene was amplified by RT-PCR and sequenced,and the SFRP1 gene of buffalo was compared with different species by bioinformatics online analysis softwares,the phylogenetic tree was constructed,and the physicochemical properties,signal peptide,and transmembrane structure of SFRP1 protein were predicted.The obtained target gene was connected to pCMV-HAhyPBase-mcheery vector and transfected into buffalo granulosa cells.The fluorescence and gene expression were detected after transfection.The expression of SFRP1 gene in different tissues of buffalo was detected by Real-time quantitative PCR.[Result]The CDS region of SFRP1 gene in buffalo was 927 bp,encoding 308 amino acids.Multiple sequence comparison results showed that the amino acid sequence of SFRP1 gene similarity between buffalo and Bos taurus,Bos mutus,Capra hircus,Orcinus orca,Pan troglodytes,Vulpes lagopus,Felis catuswas,Homo sapiens and Mus musculus were 100％,100％,99.65％,98.58％,98.23％,98.23％,98.58％,98.23％and 96.45％,respectively.There were CRD_FZ,NTR_like and DUF3367 domains.The nucleotide sequence of SFRP1 gene similarity between buffalo and Bos taurus,Ovis aries,Capra hircus,Bos mutus,Bison bison bison,Cervus elaphus,Camelus ferus,Sus scrofa and Homo sapiens were 97.3％,95.9％,95.8％,94.5％,94.2％,93.7％,80.4％,78.5％and 77.3％,respectively.Phylogenetic tree analysis showed that buffalo,Bos taurus,Bos mutus,and Bison bison bison converged into one group.Bioinformatics analysis showed that SFRP1 protein was alkaline and unstable.Signal peptide existed at amino acids 1-15,which was a secreted protein with a transmembrane structure and was mainly located outside the cell.There were 21 phosphorylation sites and 8 O-glycosylation modification sites.The secondary structure was mainly alpha-helix,extended chain and random coil.The tertiary structure of SFRP1 protein in buffalo,Bos taurus and Homo sapiens was highly similar.pCMV-mcheery-SFRP1 eukaryotic expression vector was successfully constructed and transfected into buffalo granulosa cells.After transfection 72 h,the expression of SFRP1 gene in pCMV-mcheery-SFRP1 recombinant plasmid group was extremely significantly higher than that in control group(P＜0.01).Real-time quantitative PCR result showed that SFRP1 gene was expressed in different tissues of buffalo,and the expression level in spleen was the highest,which was extremely significantly higher than that in other tissues(P＜0.01).[Conclusion]SFRP1 gene was highly conserved in different species and in the process of genetic evolution,and was widely expressed in different tissue of buffalo.The results laid a foundation for future research for the function and molecular mechanism of SFRP1 gene in the growth and development of buffalo.