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摘要：Akkermansia muciniphila偏好定殖于粘蛋白丰富的肠道黏液层中，能利用粘蛋白作为唯一的碳氮源，产生寡糖、短链脂肪酸等降解产物，为宿主提供能量的同时还可以调控脂质代谢、糖代谢和免疫反应等生物学过程以促进人体肠道健康。但A. muciniphila降解粘蛋白的相关基因功能还未全部注释，代谢通路还不清楚。本文先通过绘制生长曲线图和分析扫描电镜结果对该新型菌株的培养特性做进一步的了解，在全基因组测序的基础上选取未注释基因AKKGM001956，利用λ-Red同源重组技术构建Akkermansia muciniphila基因缺陷株，以研究目的基因在黏蛋白降解中的作用，为深入了解该菌代谢黏蛋白通路奠定基础。但由于仪器设备无法满足A. muciniphila严格厌氧的需求，Red系统在该菌上的应用仍有很多条件参数需要摸索，此次基因敲除的尝试未能成功。29486
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毕业论文关键词：Akkermansia muciniphila；粘蛋白；基因敲除
Functional Genes of Intestinal bacterium Akkermansia muciniphila
Abstract: Akkermansia muciniphila prefers to colonize in the mucin-rich intestinal mucus layer. The bacterium can use mucin as the sole carbon and nitrogen source to produce degradation products such as oligosaccharides and short chain fatty acids. These products can provide energy for the host while regulating the biological processes such as lipid metabolism, glucose metabolism and immune response to promote human intestinal health. However, the functional genes of A. muciniphila related to the degradation of mucin has not been fully annotated. The metabolic pathway is unclear. This paper first learned the growth characteristics of the new strain by plotting the growth curve and analyzing the results of scanning electron microscopy. The non-commented gene AKKGM001956 was selected on the basis of whole genome sequencing, and the Akkermansia muciniphila gene defective strain was constructed with the method of λ-Red homologous recombination system. This can help us to study the role of the target gene in mucin degradation, and to lay the foundation for further understanding the metabolic pathway of mucin. Unfortunately, as the equipment can not meet the strict anaerobic requirement of A. muciniphila and there are still many conditions and parameters need to be explored for the application of Red system on this bacteria, the gene knock-out attempt failed.
Key words: Akkermansia muciniphila; Mucin; Gene knock-out
目 录
摘要 3
关键词 3
Abstract 3
Key words 3
引言 3
1 材料与方法 4
1.1 质粒与菌株 4
1.2 试剂与仪器 4
1.3 A. muciniphila基本生长特性研究 5
1.3.1 生长曲线测定 5
1.3.2 电镜前处理 5
1.4 细菌DNA提取 5
1.4.1 DNA质量检测与送样 5
1.5 Akkermansia muciniphila基因缺陷株的构建 5
1.5.1 AKKGM001956基因敲除的技术路线 5
1.5.2 质粒pKD4的提取 6
1.5.3 pKD46和pCP20的提取方法参照2.5.2 6
1.5.4引物设计 7
1.5.5 打靶片段的PCR扩增 7
1.5.6琼脂糖凝胶电泳鉴定PCR产物 7
1.5.7 Akkermansia muciniphila感受态细胞的制备及质粒pKD4的转化 7
2 结果与分析 8
2.1 生长曲线和电镜图 8
2.2 DNA样品检测 9
2.2.1 浓度 9
2.2.2 DNA胶图 10
2.3 全基因组数据 10
2.3.1 Akkermansia muciniphila全基因组的一般特征 10