地黄C3H基因的克隆及生物信息学分析

香豆酸-3-羟化酶属于植物中最大的蛋白酶细胞色素P450家族之一,在植物生命活动中发挥着重要作用。为了解地黄香豆酸-3-羟化酶基因RgC3H合成毛蕊花糖苷的功能,该研究基于地黄代谢组学分析获得KEGG途径中的C3H,采用多重比对在NCBI中获得同源基因的一个保守序列,并基于该保守序列和地黄SRA数据库,采用电子克隆和RT-PCR克隆技术获得地黄C3H基因全长CDS(RgC3H),对其进行生物信息学分析。结果表明:RgC3H基因全长为1 530 bp,且编码一个含509个氨基酸、分子量为57.91 kD、无信号肽的蛋白质; 基于氨基酸序列的结构分析显示,RgC3H有一个保守区域-P450结构域; 系统进化分析结果显示,RgC3H与芝麻和猴面花的C3H基因具有很高的同源性。上述结果为进一步研究RgC3H基因在地黄毛蕊花糖苷生物合成途径中的作用奠定了基础。

Cloning and bioinformatics analysis of C3H gene in Rehmannia glutinosa

Coumarin-3-hydroxylase belongs to the cytochrome P450 family, one of the largest protease families,and plays an important role in plants. However,its role for verbascoside biosynthesis remains deficient. In order to understand the role of P-coumarate 3-hydroxylase for verbascoside biosynthesis in Rehmannia glutinosa(RgC3H),C3H was mined from a candidate KEGG pathway based on the metabolomics analysis of R. glutinosa, and a conserved sequence of homologous gene was obtained in NCBI by multiple alignment. According to its fragment and SRA database of R. glutinosa, we cloned its full length CDS(RgC3H)by electro cloning and RT-PCR, and performed its bioinformatics analyses. The results were as follows: RgC3H gene was 1 530 bp in length and encoded a 509-amino acid protein with a molecular weight of 57.91 kD and without signal peptide; According to its amino acid sequence and structural analysis, RgC3H contained one conserved domain-cytochrome P450 domain,suggesting that it belonged to cytochrome P450 family; Phylogenetic tree analysis showed that RgC3H had high homology with the C3H genes of Sesamum indicum and Erythranthe guttata. This study laid a foundation for further research on the role of RgC3H gene in the pathway of verbascoside biosynthesis in Rehmannia glutinosa.