论文总字数：28134字

摘 要

海洋真菌裂殖壶菌的细胞内可以合成大量二十二碳六烯酸，因此裂殖壶菌可以创造极大的经济效益。人们对裂殖壶菌的研究也在不断的进行，其目的就是提高裂殖壶菌的脂肪酸产量。除了传统的发酵法和菌株驯化，基因编辑技术也得到了研究人员的广泛应用。CRISPR/Cas9技术作为新晋出现的基因编辑技术，不同于其他那些由蛋白质引导基因编辑的技术，它的特别之处在于是由RNA引导核酸酶进行特定基因编辑。又因为CRISPR/Cas9技术经济高效、操作简单，已经在多种基因编辑工作中得到了应用。

本实验的目的就是利用CRISPR/Cas9技术构建裂殖壶菌的尿嘧啶营养缺陷型菌株。pyrF基因负责编码裂殖壶菌中尿嘧啶合成的关键酶—乳清酸磷酸核糖转移酶。本实验选择了胞内表达Cas9蛋白/sgRNA的方法进行pyrF基因的敲除。主要的内容包括：（1）选择合适的启动子用以调控Cas9蛋白的表达、寻求裂殖壶菌内源性核定位信号以及Cas9表达载体的构建。（2）找出裂殖壶菌内源性启动子调控sgRNA的表达、sgRNA序列的设计以及重组质粒的构建。（3）重组质粒的转化、目标菌株的筛选、PCR验证和相关参数的测量。

最终，本实验得到两株尿嘧啶营养缺陷型的突变型菌株。将突变型菌株的生长量及糖耗量同野生型菌株相比，并没有明显的不同，表明pyrF基因的缺失和重组质粒的表达并未对裂殖壶菌的生长产生较大影响。

关键词：裂殖壶菌；CRISPR/Cas9；pyrF基因；内源性核定位信号；内源性U6启动子

Using CRISPR / cas9 technology to construct uracil deficient Schizochytrium

Abstract

Schizochytrium sp. is a kind of marine fungus with high docosahexaenoic acid. Therefore, it has high economic value. Scientists continue to study how to improve the unsaturated fatty acids production of Schizochytrium sp.. In addition to traditional fermentation and fungous domestication,the technology of gene editing has also been favored by researchers. CRISPR / cas9 is a new gene editing technology. It is different some technologies , which are protein-guided gene editing technologies. CRISPR / cas9 technology is unique in that RNA guides nuclease to edit specific genes. Because CRISPR / cas9 technology’s cost is low and operate is easy,it is in scientist’s good graces.

The purpose of this experiment is to create the uracil deficient Schizochytrium sp. by CRISPR / cas9 technology. Orotate phosphoribosyl transferase is the key enzyme of uracil synthesis in Schizochytrium sp.. pyrF gene is responsible for coding the key enzyme. In this experiment, we selected the method of expressing cas9 protein / sgRNA in vivo to knock out the pyrF gene. The main contents include: (1) Selecting suitable promoter to regulate the expression of cas9 protein. Searching for the signal of endogenous nuclear localization of Schizochytrium sp.. And construction of cas9 expression vector. (2)To find out the endogenous promoter of Schizochytrium sp.HX-308 to regulate the expression of sgRNA. Designing sgRNA sequence. And construction of recombinant plasmid. (3) Transformation of recombinant plasmid,screening of target fungus, PCR verification and measurement of related parameters.

Finally,in this experiment, two mutant fungus of uracil deficiency type were obtained. Compared with wild-type strains, the growth and sugar consumption of mutant fungus were not significantly different,this indicated that the deletion of pyrF gene and the expression of recombinant plasmid had no obvious harm to the growth of Schizochytrium sp..

Key word:Schizochytrium sp.；CRISPR/Cas9；pyrF gene；Endogenous nuclear localization signal；Endogenous U6 promoter

目录

第一章 文献综述 1

1.1 裂殖壶菌研究概述 1

1.1.1裂殖壶菌概述 1

1.1.2 裂殖壶菌的遗传操作背景 1

1.2 CRISPR/Cas9的概述 2

1.2.1 CRISPR/Cas9的研究现状 2

1.2.2 CRISPR/Cas9的作用机制 3

1.2.3 CRISPR/Cas9基因编辑技术的发展 3

1.2.4 CRISPR/Cas9技术在微藻中的研究现状 4

1.3 小结 5

第二章 实验材料与方法 6

2.1 引言 6

2.2 实验材料 7

2.2.1 实验试剂 7

2.2.2实验仪器 9

2.2.3 培养基 10

2.3 实验方法 10

2.3.1培养方法 10

2.3.2 Cas9表达载体的构建方法 11

2.3.3 sgRNA的设计和pBS-Cas9-gRNA质粒的构建 11

2.3.4 尿嘧啶营养缺陷型裂殖壶菌菌株的筛选 11

2.3.5 相关分子生物学实验方法 12

2.3.6 发酵表观数据分析 12

第三章 实验结果与讨论 14

3.1 敲除pyrF基因的载体的构建 14

3.2 重组质粒的转化、突变型菌株的筛选及PCR验证 15

3.3突变型菌株的性能考察 16

第四章 总结与展望 19

4.1 总结 19

4.2 展望 19

参考文献 20

结束语 24

第一章 文献综述

1.1 裂殖壶菌研究概述

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