摘要：目的 克隆药用植物大戟二萜类合成途径中的CYP450基因，并且进行功能分析。 方法 采取新鲜的大戟，将大戟的根、茎、叶在液氮下分别研磨。用RNA提取试剂盒提取根、茎、叶的总RNA。通过普通琼脂糖凝胶电泳检测RNA的完整性,通过分光光度法测定RNA的纯度和浓度。以2uL的总RNA为模板，将上述样本分别反转录为cDNA，设计引物，以反转录获得的cDNA为模板PCR扩增CYP450基因的cDNA序列，对扩增产物进行琼脂糖凝胶电泳、回收、克隆入载体，经过转化后送相关公司进行测序。结果 成功克隆药用植物大戟二萜类合成途径中的一个CYP450基因--贝壳杉烯氧化酶（ent-kaurene oxidase,KO）基因，并对该基因进行了生物信息分析，对药用植物大戟根、茎、叶组织中的ko基因的表达差异也进行分析。 意义 为进一步显示药用植物大戟产生毒理作用的物质合成机制提供理论根据，并为运用基因工程提高中药材质量奠定基础。74919

毕业论文关键词： CYP450；PCR；克隆；反转录；功能分析

Cloning and functional analysis of Cytochrome P450 gene of diterpene biosynthesis pathway in medicinal plants Euphorbia pekinensis

Abstract: Purpose: Cloning cytochrome P450 gene of diterpene biosynthesis pathway in medicinal plants Euphorbia pekinensis, and analyzing its function。 Methods: Taking fresh Euphorbia pekinensis root, stem and leaf, grounding them in liquid nitrogen, respectively。 The root, stem and leaf’s total RNA were extracted using RNA extraction kit。 Electrophoresis was used to detect RNA integrity, spectrophotometry was used to detecte RNA concentration and purity。 Then above-mentioned samples were respectively reversely transcripted into cDNA。 Primers were designed and synthesized。 PCR was done to amplify cDNA sequence of cyp450 gene used cDNA as template。 The amplified products were isolated by agarose gel electrophoresis, recycled, cloned and sequenced。 Results: Successfully cloned one CYP450 gene - ene kauri oxidase gene (ko) of diterpene biosythesis pathway in medicinal plant Euphorbia pekinensis, and the functional analysis of biological information to the gene were done, differential expression of ko were also analyzed in medicinal plant Euphorbia pekinensis’s root, stem and leaf。 Significance: Our study provides a theoretical basis for the further understanding mechanism of toxic synthetic in Euphorbia pekinensis, and lays a foundation for using genetic engineering to improve the quality of Chinese herbal medicine。

Key words: CYP450; PCR; Cloning; Reverse transcription; Functional analysis

目录

引言 1

1 实验材料与方法 2

1。1 实验材料与仪器 2

1。2 实验主要试剂 2

1。3 实验方法 2

1。3。1 药用植物大戟根、茎、叶总RNA提取 2

1。3。2 反转录及PCR扩增 2

1。3。3 药用植物大戟ko基因的克隆及测序 2

1。3。4 生物信息学分析 2

1。3。5 组织表达分析 3

2 实验结果与分析 3

2。1 总RNA的提取及检测 3

2。2 药用植物大戟ko基因的克隆与序列分析 3

2。3 药用植物大戟ko基因的组织特异表达 6

3 讨论与分析 7

致谢