白木香细胞色素P450还原酶基因的克隆、表达与功能鉴定

Cloning,expression and functional identification of cytochrome P450 reductase gene in Aquilaria sinensis

细胞色素P450还原酶(cytochrome P450 reductase,CPR)是细胞色素P450酶电子传递链的组成部分,在生物体内起着重要的电子传递作用。本研究从白木香(Aquilaria sinensis)愈伤组织细胞中克隆得到1个CPR基因(Ascpr1),Ascpr1含有一个2124 bp的开放阅读框,编码707个氨基酸残基,其蛋白分子质量为78.82 kD。系统进化分析显示,AsCPR1为Ⅱ型CPR蛋白,与来源于可可树(Theobroma cacao)的CPR蛋白亲缘关系较近。跨膜预测结果显示该蛋白的第52~71位氨基酸为跨膜区,在大肠杆菌Transetta(DE3)中成功表达了N-端缺失67个氨基酸残基的融合蛋白,经亲和色谱纯化后获得对细胞色素C具有还原能力的融合蛋白AsCPR1。本研究结果为进一步研究P450酶参与的白木香防御物质合成及CPR在白木香防御反应中的作用奠定基础。

Cytochrome P450 reductase(CPR)is essential for the electron transport chain of cytochrome P450s,playing an indispensable role in electron transfer in vivo.In this study,one cDNA encoding cytochrome P450 reductase(Ascpr1)was identified from the callus of Aquilaria sinensis.Ascpr1 contains an open reading frame of 2124 bp.The deduced protein is composed of 707 amino acids,with a predicted molecular weight of 78.82 kD.Phylogenetic analysis revealed that AsCPR1 is a typeⅡCPR protein closely related to the CPR from Theobroma cacao.Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region.After truncating of 67 amino acid residues from N-terminal,the truncated AsCPR1 was successfully expressed in E.coli Transetta(DE3).Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro.The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A.sinensis.