摘要对羟基苯甲酸(4-Hydroxybenzoate，4HBA)是用途广泛的有机原料，是目前使用最广泛的防腐剂。4HBA可以从石油成分中合成出来，但其工艺能耗高，污染高，产量也低。莽草酸途径是合成4HBA的重要途径，葡萄糖经过糖酵解和磷酸戊糖途径分别生成磷酸烯醇式丙酮酸（PEP）和赤藓糖-4-磷酸（E4P），两者在 3-脱氧-β-阿拉伯庚酮糖酸-7-磷酸（DAHP）合成酶的催化下生成3-脱氧-阿拉伯庚酮糖酸-7-磷酸（DAHP）。该合成途径分析中发现一个关键基因GL003701，可能与其转化有关。本研究通过无缝克隆将其转入大肠杆菌，使其高效表达，提取粗酶，并对其进行酶活性测定，从而为4HBA的高效合成奠定基础。69841

该论文有图6幅，表1个，参考文献12篇。

毕业论文关键词：对羟基苯甲酸  莽草酸途径  无缝克隆

Characterization of 3-deoxy-β-arabinoheptulosonate-7-phosphate(DAHP) synthetase of Microbulbifer sp.

Abstract 4-Hydroxybenzoate(4HBA) is an important chemical compound , which is used widely as preservatives in the current. 4HBA is coming from the oil compound by the chemical methods, which consume a lot of energy with low production efficiency and cause a serious pollution problem.The shikimate pathway is an important pathway to synthetize 4HBA.Glucose are generated phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) through glycolysis solution and the pentose phosphate pathway.These two chemicals are generated 3-deoxy-β-arabinoheptulosonate-7-phosphate(DAHP) by the catalysis of 3-deoxy-β-arabinoheptulosonate-7-phosphate(DAHP) synthetase. A key gene,GL003701,is found in the synthetic pathway analysis, which is related to transformation.Through the seamless cloning,this study expressed GL003701 by transforming into E. coli, and carried out an enzyme assay,which can lay the foundation for the efficient synthesis of 4HBA.

Key words: 4-Hydroxybenzoate  shikimate pathway  seamless cloning

目录

摘要 Ⅰ

Abstract Ⅱ

目录 Ⅲ

图清单 Ⅴ

表清单 Ⅴ

引言 1

1.材料 2

1.1 菌株和质粒 2

1.2 分子生物学相关试剂 2

1.3 普通试剂 2

1.4 缓冲液和溶液 2

1.5仪器设备 2

2.方法 4

2.1相关溶液制备 4

2.2 CaCl2法制备JM109感受态 5

2.3 SDS碱裂解法制备质粒 6

2.4引物贮存溶液配置方法 6

2.5 GL003701基因分析 6

2.6构建表达载体 6

2.7粗酶的活性测定 9

3.结果与分析 11

3.1 GL003701基因分析 11

3.2引物设计 11

3.3 PCR扩增 12

3.4重组质粒筛选 13

3.5重组载体酶切验证 13

3.6基因测序 15

3.7粗酶的表达 16