摘要：本次实验的主要目的是克隆二萜类生物合成途径中的一个CYP450基因ent-kaurene oxidase基因（kao），并用生物信息学软件对这个基因进行功能分析。依照植物RNA提取试剂盒的提取步骤，提取药用植物大戟根、茎和叶的总RNA，反转录成cDNA，设计引物PCR扩增kao，克隆kao基因并测序，利用生物信息学软件分析kao基因的功能。利用ClustalW软件对蓖麻、麻疯树、胡杨等相关基因的氨基酸序列与药用植物大戟kao基因的氨基酸序列进行比对，结果它们之间的同源性与BLAST结果一致。进化树分析结果表明药用植物大戟与蓖麻Ricinus communis (XP_002526524)、麻风树Jatropha curcas (XP_012083781)、胡杨Populus euphratica (XP_011013095)、麻风树Jatropha curcas (XP_012084984)、胡杨Populus euphratica (XP_011010024)优先聚类。与其他植物如葡萄、梨、雷蒙德氏棉等进化距离较远。这与植物种属之间的自然进化关系相一致。组织表达分析结果表明该基因在根中没有表达。本研究为进一步揭示药用植物大戟毒性物质合成机制及利用基因工程提高中药材品质奠定基础。69055

毕业论文关键字：药用植物、大戟、CYP450、RNA提取、功能分析

Cloning and functional analysis of CYP450 gene of diterpene biosynthesis pathway in medicinal plant Euphorbia pekinensis

Abstract: The main purpose of this study is to clone a CYP450 gene-- ent-kaurene oxidase gene (kao), which was in the diterpene biosynthese pathway, and use bioinformatics software to analysis the function of this gene kao. According to the plant RNA Extraction Kit extraction steps, Total RNA of medicinal plant Euphorbia pekinensis’s root, stem and leaf were extracted, and were reverse transcripted into cDNA. PCR primers were designed to amplify and clone kao gene, and then this gene was sequenced. Bioinformatics software was used to analysis kao’s function. ClustalW software was used to do multiple alignment, and Euphorbia pekinensis’s kao with Ricinus communis, Jatropha curcas, Populus euphratica and others related sequences of amino acids were compared. The results are consistent with the blast results. The kao genes of Medicinal plant Euphorbia pekinensis and Ricinus communis (XP XP_002526524), Jatropha curcas (XP XP_012083781), Populus euphratica (XP XP_011013095), Jatropha curcas (XP XP_012084984), Populus euphratica (XP XP_011010024) are in the preferentially to get together. And other plants such as Vitis vinifera, Pyrus x bretschneideri, Gossypium raimondii and other’s evolutionary distance are far. This is consistent with the natural evolutionary relationship in plant species. Tissue expression analysis revealed that kao gene doesn’t expressed in roots. This study lays the foundation to further reveal the mechanism of medicinal plant Euphorbia pekinensis toxic substances synthesis and use genetic engineering to improve quality of traditional Chinese medicine.

key word：Medicinal plant, Euphorbia pekinensis, CYP450, RNA extraction, Functional analysis

目录

引言 4

1 材料与方法 5

1.1 材料 6

1.2 主要试剂 6

1.3 方法 6

1.3.1 药用植物大戟根、茎、叶总RNA提取 6

1.3.2 反转录及PCR扩增 6

1.3.3 药用植物大戟kao基因的克隆及测序 6

1.3.4 生物信息学分析 6

1.3.5 组织表达分析 7

2 结果与分析 7

2.1 总RNA的提取及检测