论文总字数：36245字

摘 要

番茄红素是一种天然色素具备较强的抗氧化能力，因此在医药工业中有重要作用。主要应用于预防和抑制肿瘤，调节胆固醇代谢和延缓衰老等方面。尽管对番茄红素的研究已经有很大发展，但是在快速合成番茄红素方面仍有很多困难。番茄红素合成过程有多种酶催化，属于复杂的多酶合成体系。根据这个特点，采用基因调控的方法，体外构建含有番茄红素合成中关键酶基因(crtE,crtB,crtI)的质粒，而后导入微生物中，发酵提取出番茄红素。

锌指蛋白是一种可以识别并结合到特定的12bp的目标DNA序列上的多肽蛋白，根据锌指蛋白的这个特性，本实验设计在番茄红素合成中关键酶基因上连接锌指蛋白。锌指蛋白可以确定合成番茄红素中关键酶的结合的专一位点。构建与锌指蛋白结合的DNA支架，最后将含有锌指蛋白的番茄红素多酶表达质粒和DNA支架一起导入大肠杆菌中，通过增加酶与酶之间接触和中间体的局部浓度来实现生产最大化。

关键词：番茄红素 多酶合成体系 基因调控 锌指蛋白

Plasmid construction of lycopene synthesis module which contains zine finger protein binding domain

Abstract

Lycopene is natural pigment,Lycopene plays an important role in pharmaceutical,due to its strong antioxidant ability.It is mainly used in the prevention and inhibition of tumor,regulating cholesterol metabolism and anti-aging,etc.Although the study of lycopene amylase has been carried out and made important progress,the rapid synthesis of lycopene in vitro still exist many difficulties.Lycopene synthesis process is a kind of complex multi enzyme synthesis system,which many enzymes involve in.Using this feature can building plasmid contains lycopene synthesis of key enzyme's gene(crtE,crtB,crtI) in vitro with the method of gene regulation. Then,lycopene gets from microbial fermentation.

A zineprotein recognize and bind to specific 12-bp target DNA sequences,based on the characterisrics of zine finger protein, in this study lycopene synthesis of key enzyme's gene link a zine protein.A zine proteinserves as an adapter for the site-specific binding of each enzyme involved in lycopeneproduction to a precisely ordered.Build DNA scaffod combined withzine finger protein ,at last finally,conbine zine finger protein’s lycopene multi enzyme expression plasmid and DNA scaffold import Escherichia coli,to increase the proximity of enzyme and the local concentration of metabolites to maximize production.

Key Words:Lycopene;Multi enzyme synthesissystem;Gene regulation;

Zine finger protein

目 录

摘要..........................................................................................................Ⅰ

Abstract..................................................................................................Ⅱ

第一章 文献综述......................................................................................1

1.1番茄红素..............................................................................................................1

1.1.1番茄红素的概况和生理功能.......................................................................1

1.1.2番茄红素的生产方法...................................................................................1

1.1.3番茄红素的生物合成....................................................................................2

1.1.4番茄红素合成中的关键酶...........................................................................4

1.2番茄红素合成中的基因调控..............................................................................5

1.3多酶合成体系......................................................................................................6

1.3.1多酶合成体系的应用...................................................................................6

1.3.2多酶合成体系的组装方式...........................................................................7

1.4研究任务、目的和意义......................................................................................8

1.4.1研究任务.......................................................................................................8

1.4.2目的和意义...................................................................................................9

第二章 实验材料、方法及实验流程......................................................10

2.1实验材料.............................................................................................................10

2.1.1实验仪器......................................................................................................10

2.1.2实验试剂......................................................................................................10

2.1.3实验菌株与质粒..........................................................................................11

2.1.4培养基的配方及缓冲液的配制.................................................................12

2.2实验方法............................................................................................................12

2.2.1琼脂糖凝胶电泳.........................................................................................12

2.2.2PCR引物设计..............................................................................................13

2.2.3单链变成双链.............................................................................................13

2.2.4酶切与连接.................................................................................................14

2.2.5胶回收.........................................................................................................14

2.2.6转化.............................................................................................................14

2.2.7质粒快检......................................................................................................15

2.3实验流程............................................................................................................16

2.3.1支架的构建流程.........................................................................................16

2.3.2含锌指蛋白基因的目的基因构建流程.....................................................18

第三章 实验结果与分析......................................................................29

3.1实验结果.............................................................................................................28

3.1.1DNA支架S123/S132的构建.....................................................................28

3.1.2构建pET22b-ADB1 crtE...........................................................................31

3.1.3构建pET22b-ADB1 crtE-ADB2 crtB......................................................33

3.1.4构建pET22b-ADB1 crtE-ADB2 crtB-ADB3 crtI...................................35

3.1.5大肠杆菌中提取双质粒..............................................................................37

3.2结果分析...........................................................................................................38

第四章 结论与展望................................................................................39

4.1结论....................................................................................................................39

4.2展望....................................................................................................................39

参考文献..................................................................................................41

致谢..........................................................................................................44

1. 文献综述
   1. 番茄红素
      1. 番茄红素的概况及生理功能

番茄红素是一类具有抗氧化活性的重要萜.类化合物。番茄.红素命名的由来：首先Hartsen从番茄中提取出一种深红色的晶体，随后Schunch将这种提取物称为番茄红素。番茄红素在自然.界存在十分普遍，尤其是成熟的番茄中，其次是一些蔬菜后者水果中^[1]。其有“植物黄金”，“藏在西红柿里的黄金”等美称^[2]。

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