摘要：细胞色素P450 还原酶(cytochrome P450 reductase, CPR)具备给氧化系统提供电子的作用，因此在生物体的氧化还原反应中扮演着限速酶的角色。药用植物大戟的主要活性成分为萜类化合物，细胞色素P450参与了萜类化合物的合成，因此研究药用植物大戟CPR具有重要的意义。本研究克隆了药用植物大戟细胞色素P450还原酶基因的开放阅读框序列，序列全长2115 bp，编码704个氨基酸序列。序列比对发现药用植物大戟CPR与麻疯树、巴豆、可可和棉花等植物的CPR同源性较高，进化树分析结果表明药用植物大戟CPR序列的进化与植物种属之间的自然进化关系相一致。组织特异表达分析结果表明药用植物大戟CPR基因在根中表达量较高，茎中其次，叶中表达量相对较低。因为CPR基因参与药用植物大戟萜类生物合成，因此本研究为更进一步研究药用植物大戟萜类活性成分的生物合成奠定基础。69052

毕业论文关键词：细胞色素P450还原酶；克隆；PCR；生物合成

Cloning and functional analysis of cytochrome P450 reductase gene of medicinal plant Euphorbia pekinensis

Abstract: Cytochrome P450 reductase (cytochrome P450 reductase, CPR) has the function of providing electron for the oxidation system, thus served as the limited enzyme of the oxidation reduction reactions in living organisms. The active component of medicinal plant Euphorbia pekinensis is terpenoids, and CYP450s are involved in terpenoids biosynthesis pathway. Thus it is of great significance to study the CPR gene of Euphorbia pekinensis. In this study we cloned the ORF of cytochrome P450 reductase gene (cpr), the ORF sequence of cpr is 2115 bp, coding a 704 amino acid sequence. Sequence alignment in NCBI showed that CPR homology in Euphorbia pekinensis and Jatropha curcas、Croton stellatopilosus、Theobroma cacao、Gossypium hirsutum is higher. The evolutionary tree analysis results showed that the medicinal plant Euphorbia pekinensis’s CPR sequence evolution is consistent with the natural evolution among plant species. Tissue specific gene expression analysis results showed that Euphorbia pekinensis cpr gene expressed in root is higher, and second is stem, cpr gene expression in leaf is relatively low. CPR gene is involved in terpenoids biosynthesis in Euphorbia pekinensis, so this study laid the foundation for futher studying the terpenoids biosynthese in Euphorbia pekinensis.

Key words: cytochrome P450 reductase; cloning; PCR; biosynthesis

目录

引言 1

1材料与方法 2

1.1实验材料 2

1.2 主要试剂 2

1.3 主要仪器 3

1.4 操方作法 3

1.4.1 药用植物大戟根、茎、叶总RNA提取 3

1.4.2 反转录及PCR扩增 3

1.4.3 药用植物大戟cypr基因的克隆及测序 3

1.4.4 生物信息学分析 3

1.4.5 组织表达分析 4

2 结果与分析 4

2.1 总RNA的提取及检测 4

2.2 药用植物大戟cypr基因的克隆与序列分析 4

2.3 药用植物大戟cypr基因的组织特异表达 8

3 讨论 9

致 谢 9

参考文献 10