滇龙胆中细胞色素P450还原酶基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Cytochrome P450 Reductase Gene in Gentiana rigescens

细胞色素P450还原酶在体内可催化许多反应,通过FAD和FMN辅基将NAD(P)H的电子传递至细胞色素P450酶,从而使细胞色素P450酶同底物产生氧化还原反应,起到电子供体的作用。本研究对药用植物滇龙胆(Gentiana rigescen Franch.)的细胞色素还原酶基因GrCPR1进行克隆、生信分析和异源表达。结果表明,GrCPR1基因cDNA全长2078 bp,相对分子质量77.10 kD,含691个氨基酸,等电点为5.01,含73个极性氨基酸残基,104个非极性氨基酸残基,平均疏水性为-0.330,脂溶系数为82.56,分子式C3449H5349N917O1046S22,半衰期为30 h。不稳定指数42.25,属于不稳定亲水蛋白。GrCPR1蛋白为非分泌蛋白,不具有信号肽识别功能。第27~46位氨基酸为该蛋白跨膜区,同时亚细胞定位预测分析发现GrCPR1蛋白位于内质网上。CPR是为细胞色素单加氧酶CYP的催化过程提供电子,在P450系统中起重要作用。本研究结果可以为今后研究龙胆苦苷的生物合成途径的CYP基因功能提供重要的数据支持。

The cytochrome P450 reductase can catalyze many metabolic reactions in vivo. It can transfer electrons from NAD(P)H to the cytochrome P450 enzyme through FAD and FMN auxiliary groups which makes cytochrome P450 enzyme react with substrate to produce redox reaction and act as electron donor. In this study,the cytochrome reductase gene GrCPR1 of the medicinal plant Gentiana rigescen Franch. was cloned, analyzed and heterologously expressed. The results showed that the cDNA total length of GrCPR11 was 2 078 bp. The relative molecular weight was 77.10 kD. It contained 691 amino acids with an isoelectric point of 5.01, 73 polar amino acid residues and 104 non-polar amino acid residues, with an average hydrophobicity of-0.330, a liposolubility coefficient of 82.56, a molecular formula of C3449 H5349 N917 O1046 S22, and a half-life period of 30 h. The instability index of Gr CPR1 protein was 42.25, which belonged to unstable hydrophilic protein. GrCPR1 was a non-secretory protein and didn’t have the function of signal peptide reco gnition. Amino acid at positions 27 to 46 were the transmembrane region of the protein, and subcellular localization prediction analysis showed that the GrCPR1 was located in the endoplasmic reticulum. CPR provides electrons for the catalytic process of cytochrome monooxygenase CYP and plays an important role in the P450 system. The results provide important support for the study of the function of CYP gene in the biosynthesis pathway of Gentiana rigescen.