论文总字数：22087字

摘 要

在裂殖壶菌中，酰基转移酶（Acyltransferase，AT）位于聚酮合酶ORFB基因簇的第二个域，还有一个特殊的结构GxSxG，这个特殊的结构可能与PUFAs的释放有关。但是，多不饱和脂肪酸从聚酮合酶上释放的机制任然是不清晰的。在哺乳动物系统中，最终的产物是通过硫酯酶释放为游离脂肪酸的，但是在裂殖壶菌中并没有发现硫酯酶，裂殖壶菌的AT酶可能扮演者硫酯酶的功能。另外，许多次级代谢产物，如洛伐他汀，角鲨抑素和伏马菌素等都是通过AT域释放的。但是，在裂殖壶菌中AT是否负责脂肪酸的释放以及是否决定脂肪酸种尚不清晰。为了研究酰基转移酶对最终PUFA产品种类的影响，以及其在不饱和脂肪酸合成过程中扮演的重要角色，获得该酶各个催化模块的蛋白至关重要，

因此本论文旨在通过PCR方法扩增裂殖壶菌酰基转移酶（AT）基因，并将其与载体pET28a接连获得pET28a AT。将质粒通过化学转化法转入大肠杆菌DH5α以及BL21感受态细胞中，获得AT基因工程菌株，进行诱导，通过蛋白电泳验证AT酶是否成功表达。

关键词： 裂殖壶菌 酰基转移酶（AT） pET28a PUFA

Construction of genetic engineering Escherichia coli. for

Acyltransferase production

Abstract

In the Schizochytrium sp,Acyltransferase  located in  the second domain of polyketone synthase ORFB gene cluster .It also has a special structure GxSxG, this special structure may be associated with the release of PUFAs.However, the mechanism for releasing polyunsaturated fatty acids from the polyketase is not clear.In the mammalian system, the final product was released as free fatty acid by thiesterase enzyme, but no thiesterase was found in the Schizochytrium sp.The AT of the Schizochytrium sp may play a role in the function of thiesterase. In addition, a number of secondary metabolites, such as lovastatin, squalin and vomarycin, were released through the AT domain.However, it is not clear whether AT is in charge of the release of the fatty acid and the determine the fatty acids .In order to study the acyltransferase impact on final PUFA kinds of products and the  important role  in the synthesis of unsaturated fatty acid,to explore the specific enzyme catalytic mechanism, different catalytic module of this enzyme are obtained protein is very important.

Therefore, this paper aims to construct genetic engineering E. coli. Firstly, the AT gene fragments were respectively amplified from Schizochytrium sp. by PCR, and ligated into pET28a carrier.. Then, the plasmids are transformed to into E. coli BL21 to generate AT production strains. The finally confirm engineering strains whether to express successfully by a protein electrophoresis tests.

Key Words: Schizochytrium sp.;Acyltransferase(AT);pET28a;PUFA

目 录

摘要 I

Abstract II

第一章 文献综述 5

1.1 DHA概述 5

1.2 裂殖壶菌概述 5

1.3 裂殖壶菌脂肪酸合成途径 6

1.4 酰基转移酶（AT）概述 9

1.4.1酰基转移酶简介 9

1.4.2聚酮合酶研究现状 10

1.5 酰基转移酶（Acyltransferase，AT）概述 10

1.6 总概述 10

第二章 实验材料与设备 6

2.1 实验材料 6

2.1.1 实验仪器 6

2.2 培养基 8

2.2.1 裂殖壶菌种子培养基： 8

2.2.2 LB培养基： 9

2.2.3 Kan抗性的固体培养基 9

2.3菌种保藏 9

2.4 实验中涉及的引物 10

2.5基因操作相关实验方法 10

第三章 实验方法 11

3.1 目标载体的构建 11

3.1.1 1500-AT与pET28a重组质粒的构建 11

3.1.2 1947-AT与pET28a重组质粒的构建 14

3.2 蛋白诱导表达 16

第四章 结果与讨论 17

4.1 裂殖壶菌1500-AT pET28a载体验证 17

4.1.1裂殖壶菌1500-AT片段的验证 17

4.1.2 1500-AT片段与pET28a载体的构建验证 17

4.2裂殖壶菌1947-AT pET28a载体验证 18

4.2.1裂殖壶菌1947-AT片段的验证 19

4.2.2 1947-AT片段与pET28a载体的构建验证 19

4.3 目标产物蛋白的诱导 20

4.3.1 1500-AT pET28a、1947-AT pET28a基因工程菌的沉淀蛋白电泳图 20

4.3.2 1500-AT pET28a、1947-AT pET28a基因工程菌的上清蛋白电泳图 21

第五章 结论与展望 22

5.1 结论 22

5.2展望 22

参考文献 23

致谢 26

第一章 文献综述

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