摘要苦蘵(Physalis angulata)是我国一种传统的药用植物，其为茄科植物苦蘵的全草、灌木、小乔木。浆果或蒴果，约85属3000余种。广泛分布于全世界温带及热带地区，南美洲为最大的分布中心，种类最多。作为中医药用植物苦蘵具有清热，利尿，解毒，消肿功效。51722

以苦蘵作为研究对象，查尔酮关键基因包括了异构酶基因和合成酶基因。查尔酮异构酶与类黄酮物质的合成有着密切的关系，其转基因表达程度的研究对于提高植物类黄酮含量起到重要作用。查尔酮合成酶是败酱科植物合成途径中主要的酶，研究查尔酮合成酶编码基因的克隆可以影响查尔酮酮化合物在苦蘵中的产量。

本实验我们通过TA克隆获得了我们需要的查尔酮合成酶基因和查尔酮异构酶基因的全长序列，并且构建了两个基因的GFP荧光载体。同时我们利用苦蘵叶肉细胞，建立了蛋白瞬时表达体系，为在本物种中研究蛋白的定位情况提供了可能。

毕业论文关键字：苦蘵；查尔酮；基因克隆；荧光载体

Abstract Physalis angulate is a traditional medicinal plant in China, its for solanaceae plant bitter all grass, shrubs, small trees ,Berries or capsule, about 85 genera and 3000 species. It widely distributed all over the world temperate and tropical regions, South America's largest distribution center, most species. As a traditional Chinese medicine with plants bitter  has clear heat, diuresis, detoxicating, detumescence effect.

With bitter as the research object, the key genes including chalcone isomerase gene and synthetase gene. Isomerase chalcone and flavonoids substances has close relation with the synthesis of the genetically modified (gm) expression level of study play an important role for the improvement of plant flavonoids content. Synthetase is routed chalcone dressing plant main enzymes in the synthesis pathway, synthase gene encoding chalcone research cloning can affect ketone compounds in bitter  chalcone in production.

This experiment we obtained we need through TA cloning synthetase gene and chalcone isomerase chalcone gene sequence, and constructed two gene GFP fluorescence carrier. At the same time, we used the bitter  mesophyll cells, protein transient expression system is established, for the localization of protein research in this species.

Key words: Physalis angulata; Charl; gene clone; fluorescent carrier

目录

引言 6

1.研究材料 8

1.1实验材料 8

1.2化学试剂 8

1.3培养基 8

1.4主要仪器 8

2.研究方法 9

2.1苦蘵不同部位总RNA提取（Trizol法） 9

2.2 RNA质量检测 9

2.3 cDNA模板的合成 9

2.4 PCR扩增目的基因 10

2.5 目的片段的纯化回收 11

2.6目的片段与T-载体的连接 11

2.7转化与筛选 12

2.7.1重组质粒的提取： 12

2.8质粒的鉴定及测序 13

2.9.1农杆菌转化侵染 14

3. 结果与分析 14

3.1 苦蘵不同部位总RNA提取 14

3.2 cDNA质量检测 15