玉米螟细胞色素P450家族CYP6AE25的克隆及序列分析

Cloning and Sequence Analysis of Cytochrome P450 CYP6AE25 Gene from Ostrinia furnacalis

昆虫细胞色素P450是一类在生物代谢中起重要作用的基因家族,承担着许多重要的生理功能,研究其对植物次生物质代谢和杀虫剂的解毒等具有重要的意义。采用同源克隆方法与RACE技术克隆玉米螟细胞色素P450全长基因,并运用生物信息学软件,对其蛋白的理化性质、结构组成、信号肽、跨膜结构域、二级结构、三级结构与功能结构域等作了较为详细的分析与预测。结果表明,利用简并引物扩增出了玉米螟细胞色素P450基因的中间片段,通过RACE技术获得玉米螟P450基因家族一员全长序列,即编码523个氨基酸残基的2315bp的全长cDNA,命名为OfP450(GenBank登录号:EU807990)。国际P450命名委员会将其定为CYP6AE25。生物信息学结果,从CYP6AE25蛋白的理化、结构与功能等作了更为深入的数据分析。为进一步研究其高级结构与功能的关系提供理论依据。CYP6AE25蛋白与其他P450分子进化树分析结果显示,与昆虫CYP6家族同源性较高,与CYP其他家族的遗传距离较远,同源性较低。

Insect cytochrome P450 is a kind of gene family that plays important roles in biological metabolism and performs many important physiological functions, including, the plant secondary metabolism and insect resistance to insecticides. The cDNA encoding the cytochrome P450 protein was isolated from Ostrinia fumacalis larva midgut by homology-based cloning and RACE technology. The sequences was analyzed and pridicted through the bioinformatics method which analyzed physical and chemical properties, composition, signal peptide, transmembrane domain, secondary structure, tertiary structure and functional structure domain. The results showed that the P450 middle fragment is cloned by a pair of degenerate primers designing based on the reported sequences of conservative amino acid regions of P450 protein family in insects. Then a 2315 bp full-length of Ostrinia fumacalis P450 gene is obtained （GenBank accession no. EU807990）, encoding 523 amino acid residues. The sequence of Of P450 is named as CYP6AE25 by the International P450 Nomenclature Committee. Sequnence analysis showed that the research made a more detailed analysis and forecast for CYP6AE25 from the aspects of properties, structure and function. It provides a theoretical basis to study between the relationship structure and function. Molecular evolutionary tree analysis revealed that CYP6AE25 protein and other insect family P450 have higher homology, compared with the other CYP family, farther genetic distance, less homologous.