Cloning,expression and functional identification of cytochrome P450 reductase of Bufo gargarizans
Objective:To clone cytochrome P450 reductase gene from the liver tissue of Bufo gargarizans Cantor(BgCPR,GenBank accession number:OQ572435)for bioinformatics analysis and functional verification.Methods:BgCPR gene sequence was mined from the transcriptome database of Bufo gargarizans.Specific primers at both ends of BgCPR gene were designed using SnapGene,and cDNA reverse transcribed from total RNA of liver tissue of Bufo gargarizans was used as a template for PCR amplification.Bioinformatics was applied to analyze the physicochemical properties and the spatial structure of BgCPR protein,and the phylogenetic tree was constructed.On the basis of the CRISPR/Cas9 gene editing system,the expression cassette of BgCPR gene was integrated into Saccharomyces cerevisiae genome through homologous recombination,and transformant microsomes were prepared by polyethylene glycol method to verify the reductive activity to cytochrome C.Results:A total length of 2 043 bp cDNA of BgCPR gene was cloned,encoding 680 amino acids with the relative molecular mass 77 852 Da.Structural prediction showed that BgCPR protein was a non-secreted protein.There was a transmembrane helical domain located between amino acids 24 and 46 of the protein.Subcellular localization prediction showed that the protein located on the endoplasmic reticulum.In vitro enzyme activity experiment proved that it has the electron transferring function.Conclusions:In this study,the BgCPR gene of Bufo gargarizans was cloned,and BgCPR S.cerevisiae engineered cells expressing bioactivity was constructed,which laid a foundation for further study on the function of bufo C YP450 gene and even analyzing the biosynthetic pathway of bufadienolides.
万方数据
维普
