论文总字数：20316字

摘 要

21世纪以来，发酵技术越来越多的应用在了工业生产的各个方面。多糖则是发酵产业的一大产物，被广泛的用在食品、化妆品等大型产业链中。微生物多糖（又称胞外多糖）是一类离子型或非离子型的水溶性生物高分子，自1880年葡聚糖合成以来，微生物多糖由于其独特结构和物理性质,在食品、医药等行业中具有较高的应用价值，已被用作胶粘剂，吸附剂，润滑剂，土壤改良剂，化妆品，药物载体和高强度结构材料等，胞外多糖相比如天然植物多糖，更加的可控，不受一些不可控因素的影响。所以，如何更大化的提高多糖产量，降低发酵的成本，是本实验想要达到的目的。据悉，发酵工业中存在的高资源和能源消耗是限制其大规模发展的主要原因之一，而这一问题在微生物胞外多糖发酵生产中尤其突出。胞外多糖生成往往伴随着类胡萝卜素此类色素的生成，提取时需要大量乙醇来分离，同时也会对下游造成污染，提高了成本，因此本实验旨在从基因敲除角度来解决这个问题，降低工业成本。我们使用crisper/Cas9技术，先用PCR技术对DNA双链进行定位、切割。再对质粒进行PCR并将其与前者同源重组，然后经过热激转化并利用大肠杆菌培养质粒，再接着提取质粒和制备感受态，将质粒敲入，培养，得到我们要的基因编辑过后的菌。在得到菌之后，我们将菌放在suc培养基中培养，最终得到并测定它的产胞外多糖的能力。实验最终得出，CrtB基因能够有效的被人为敲除，并且不会影响细菌产多糖的能力。

关键词：胞外多糖 基因编辑 PCR 类胡萝卜素

Study on the effect of nx-11 pigment gene knockout on polysaccharide yield of panicularia camellia l

Abstract

Since the 21st century, fermentation technology has been more and more applied in all aspects of industrial production. Polysaccharide is a major product of fermentation industry, which is widely used in food, cosmetics and other large industrial chains. Microbial polysaccharides (also called extracellular polysaccharide) is a kind of water soluble ionic and nonionic biological macromolecule, glucan synthesis since 1880, microbial polysaccharide due to its unique structure and physical properties, in the food, pharmaceutical and other industries with high application value, has been used as adhesive, adsorbent, lubricant, soil conditioner, cosmetics, such as drug carrier and high strength structural materials, compared to extracellular polysaccharides such as natural plant polysaccharides, more controllable, not influenced by some uncontrolled factors. Therefore, how to increase the yield of polysaccharide and reduce the cost of fermentation is the purpose of this experiment. It is reported that the high resource and energy consumption in the fermentation industry is one of the main reasons to limit its large-scale development, and this problem is particularly prominent in the fermentation production of microbial exopolysaccharides. The production of exopolysaccharides is usually accompanied by the production of pigments such as carotenoids. A large amount of ethanol is needed to separate exopolysaccharides, which also causes pollution to the downstream and increases the cost. Therefore, this experiment aims to solve this problem from the perspective of gene knockout and reduce the industrial cost. Using the crisper/Cas9 technology, we first used PCR technology to locate and cut the DNA double strand. Then PCR was performed on the plasmid and homologous recombination was performed with the plasmid. Then the plasmid was transformed by heat shock and cultured by escherichia coli. Then the plasmid was extracted and the sensitized state was prepared. After obtaining the bacteria, we cultured the bacteria in suc medium, and finally obtained and measured its capacity to produce exopolysaccharides. The result was that the CrtB gene could be effectively knocked out without affecting the bacteria's ability to produce polysaccharides.

Key Words: Extracellularpolysaccharide；gene editing ；PCR ；carotenoids

目录

摘要 I

第一章 文献综述 1

1.1微生物多糖概述 1

1.2 类胡萝卜素概述 2

1.2.1 类胡萝卜素基本概况 2

1.2.2 类胡萝卜素的结构 2

1.2.3 类胡萝卜素的合成途径 3

1.3 CRISPR/Cas9基因编辑技术 4

1.3.1 CRISPR/Cas9技术简介 4

1.3.2 CRISPR/Cas9技术的发展历程 5

1.3.4CRISPR/Cas9技术的应用 7

第二章 实验材料与方法 8

2.1实验仪器和材料 8

2.1.1实验试剂以及仪器 8

2.1.2分子生物试剂（盒） 9

2.1.3培养条件 9

2.2P. alhagi NX-11 的基因组的DNA 提取 9

2.3 PCR反应 9

2.4 质粒提取、胶回收 10

2.5连接体系 11

2.6 大肠杆菌感受态的制备及热激转化 11

2.7 P. alhagi NX-11电转感受态制备与电转 12

2.8 P. alhagi NX-11 突变体的构建 13

2.9 NX-11胞外多糖含量测定 13

第三章实验结果与讨论 15

3.1 pTarget-crtB质粒的构建 15

3.2 pCas的转化 16

3.3 crtB敲除菌株的构建 16

3.4 crtB敲除对胞外多糖产量的影响 17

第四章结论与展望 19

4.1结论 19

4.2展望 19

参考文献 20

致谢 22

第一章 文献综述

1.1微生物多糖概述

发酵工业是现代工业的重要组成，其产品广泛应用各大行业，并支撑着高额的工业产值，是相关企业发展的基础。然而，随着水资源流失、环境污染、粮食匮乏等问题变得逐渐严重，发酵工业能耗大、排污多、与人分粮的缺陷也逐渐显露出来。因为在工业发酵的环境下,微生物面临非常复杂的生理化学应用和环境条件的胁迫^[1,2],如高温、pH大小、高粘度等，为了维持微生物所需的生存与生产条件，需要分别采用循环冷却、碱（或酸）物质中和，增加通气和搅拌等措施，但是这些措施会产生大量的能耗和下游污染，因此我国发酵行业能耗较国际先进水平高40%，水耗高55%，发酵行业废水排放达80亿立方米/年、COD排放500万吨/年，分别占工业排放总量10%、20%。此外,我国的现有耕地面积虽然已经占到了2010年世界人均现有耕地面积的百分总量7%,但土地生产和农业养育的3000万农业劳动人口却仅仅约占了2010年全世界我国现有3000万农业劳动人口人均产出产值总额的21%,而且随着人地比例越发不平衡，工业大量使用粮食加剧了粮食短缺的局面^[3]。以上问题在微生物多糖——威兰胶的发酵生产中尤为突出。

请支付后下载全文，论文总字数：20316字