中华大蟾蜍细胞色素P450还原酶基因克隆、表达与功能鉴定

Cloning,expression and functional identification of cytochrome P450 reductase of Bufo gargarizans

目的:从中华大蟾蜍(Bufo gargarizans Cantor)肝脏组织中克隆细胞色素P450还原酶基因(BgCPR,GenBank登录号:OQ572435)进行生物信息学分析,并进行功能验证。方法:基于中华大蟾蜍转录组数据库挖掘BgCPR基因序列,使用SnapGene软件设计BgCPR基因两端特异性引物,以蟾蜍肝脏组织总RNA逆转录成的cDNA为模板进行PCR扩增。利用生物信息学分析BgCPR蛋白理化性质、空间结构并构建系统进化树;基于CRISPR/Cas9基因编辑系统将蟾蜍BgCPR基因表达框经同源重组整合进入酿酒酵母基因组,利用聚乙二醇法制备转化子微粒体,验证其对细胞色素C的还原功能。结果:克隆得到全长2043 bp的BgCPR基因cDNA,编码680个氨基酸,相对分子质量77852 Da。结构预测显示,BgCPR蛋白为非分泌蛋白;有一段跨膜螺旋区,位于蛋白的第24~46位氨基酸之间;亚细胞定位预测显示该蛋白定位于内质网上。采用体外酶活实验证明其具有传递电子功能。结论:本研究克隆得到中华大蟾蜍BgCPR基因,并构建了表达具有生物活性的BgCPR的酿酒酵母工程细胞,为进一步研究蟾蜍CYP450基因功能乃至解析蟾蜍二烯内酯类化合物的生物合成途径奠定了基础。

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 CYP450 gene and even analyzing the biosynthetic pathway of bufadienolides.