论文总字数：16808字

摘 要

裂殖壶菌可以发酵生产DHA，为了提高DHA的产量，有必要对裂殖壶菌中多不饱和脂肪酸的合成途径进行研究。在测得的转录组数据中，Δ12去饱和酶是同类酶转录水平的10倍以上。据报道，Δ12去饱和酶引入双键催化油酸形成亚油酸是生产多不饱和脂肪酸的第一步。越来越多的证据表明裂殖壶菌中存在两种合成的多不饱和脂肪酸途径：聚酮化合物合成酶（PUFA合成酶）和去饱和酶/延伸酶（标准）途径。

裂殖壶菌是研究DHA生物合成途径的主要海藻之一，因为其DHA的含量在总脂肪酸中的占比高达35%，而且在这个途径中的其他中间产物的含量是极小的，所以DHA的积累可以说明在裂殖壶菌DHA的生物合成途径中一系列酶（延长酶和去饱和酶等）在引导不同的中间产物转化为最终产物DHA的方面有不同寻常的作用，所以可以复制裂殖壶菌中不同的去饱和酶的基因并且去验证这些不同基因的功能。因时间的缘故，本实验就克隆了Δ12去饱和酶的基因并进行了功能验证。

本实验主要运用基因工程和分子生物学的实验方法，首先克隆出裂殖壶菌中Δ12 去饱和酶的基因片段，构建重组质粒，之后将验证过的重组质粒导入构建好的酵母表达载体中，诱导酵母表达载体正确表达，离心弃上清液，重悬于缓冲盐水，液氮反复冻融，离心跑SDS-PAGE进行验证是否有目的蛋白。离心得到菌体，烘干破碎做脂肪酸甲酯化，提油上气象做脂肪酸分析。比较脂肪酸气相色谱图可以大概看出酶对产脂肪酸的影响，从而以此为依据，进一步论证提高脂肪酸产量的方法。

关键词：裂殖壶菌；Δ12去饱和酶；脂肪酸

Functional Identification of Schizochytrium Δ 12 Desaturase

Abstract

Schizochytrium can ferment to produce DHA, in order to further increase the yield of DHA, it is necessary to study the synthesis pathway of polyunsaturated fatty acids in Vibrio cholerae. In the measured transcriptome data, the Δ12 desaturase is more than 10 times the transcription level of the same enzyme. It has been reported that the introduction of a double bond by a Δ12 desaturase to catalyze the formation of linoleic acid is the first step in the production of polyunsaturated fatty acids. There is increasing evidence that there are two synthetic polyunsaturated fatty acid pathways in Schizochytrium: the polyketide synthase (PUFA synthase) and the desaturase/elongase (standard) pathway.

Schizochytrium is one of the major seaweeds that study the DHA biosynthetic pathway because its DHA content accounts for up to 35% of total fatty acids, and the content of other intermediates in this pathway is minimal, so DHA The accumulation can indicate that a series of enzymes (elongation enzymes and desaturase, etc.) in the biosynthetic pathway of Schizochytrium DHA have an unusual role in guiding the conversion of different intermediates into the final product DHA, so they can replicate The genes for different desaturases in the genus of the genus and to verify the function of these different genes. For the sake of time, only the gene of Δ12 desaturase was duplicated and functionally verified.

This experiment uses genetic engineering and molecular biology experimental methods to clone the gene fragment of Δ12 desaturase in Schizochytrium，construct a recombinant plasmid, and then introduce the verified recombinant plasmid into the constructed yeast expression vector. The yeast expression vector was induced to be correctly expressed, the supernatant was centrifuged, resuspended in buffered saline, and liquid nitrogen was frozen and thawed, and centrifuged to perform SDS-PAGE to verify whether there was a protein of interest. These were obtained by centrifugation, dried and crushed to make fatty acid methyl esterification, and the oil was analyzed on the weather for fatty acid analysis. Comparing fatty acid gas chromatograms can roughly show the effect of enzymes on fatty acids production, and based on this, further demonstrates ways to increase fatty acid production.

Key Words：Schizochytrium; Δ12 desaturase; fatty acid

目录

摘要 I

Abstract II

第一章 文献综述 1

1.1 研究现状 1

1.2 裂殖壶菌 1

1.3 不饱和脂肪酸 2

1.4 Δ12去饱和酶 2

1.5 本实验的内容和意义 5

第二章 实验材料与方法 6

2.1 实验材料 6

2.1.1 实验试剂 6

2.1.2 实验仪器 7

2.1.3 菌种，质粒和引物 7

2.1.4 培养基 7

2.2 质粒的提取 8

2.3 PCR 9

2.3.1 PCR扩增 9

2.3.2 核酸验证 9

2.3.3 PCR纯化 10

2.4 质粒的重组 10

2.4.1 双酶切 10

2.4.2 胶回收 11

2.4.3 连接 11

2.5 酵母表达载体的构建 11

2.5.1 TBS（Tis-buffer saline）的制备 11

2.5.2 感受态细胞的制备 11

2.5.3 转化 12

2.6 酵母转化子的筛选 12

2.7 诱导酵母表达 12

2.8 酵母表达载体蛋白的获取与检测 13

2.9 脂肪酸分析 13

2.9.1 重组酵母培养 13

2.9.2 脂肪酸甲酯化 14

2.9.3 气相色谱 14

第三章 结果与分析 15

3.1 质粒的构建 15

3.2 酵母表达载体的构建和表达 16

3.3 脂肪酸检测 17

第四章 结论与展望 18

4.1 结论 18

4.2 展望 18

参考文献 19

致谢 22

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