肺炎病患下呼吸道及沼气发酵体系的宏基因组学研究

发布时间：2018-03-25 03:15

本文选题：宏基因组学　切入点：焦磷酸测序　出处：《中国科学院北京基因组研究所》2013年博士论文

【摘要】：作为一个新兴的研究领域,宏基因组学为微生物研究开拓了新的空间。宏基因组学凭借其高新的技术,已经渗透到众多复杂环境研究领域,例如海洋、土壤、热泉、人体口腔及胃肠道、替代能源、环境修复、农业和生物防御等。正常人体下呼吸道是一个相对封闭的无菌环境,细菌侵染下呼吸道会对人体健康乃至生命造成极大的威胁。目前人体下呼吸道微生物组研究由于受到取样方法等问题的限制,研究尚不够深入。而在人体环境之外其他封闭环境中,例如沼气发酵体系,宏基因组学研究也尚在起步阶段。沼气发酵体系宏基因组学研究,可以揭示发酵体系内菌群功能特征,对提高甲烷产气量、指导工业化生产有重要意义。本文针对人体内、外环境分别选取肺炎病患下呼吸道和沼气发酵体系为代表,优化了不同样品DNA获得方法,通过高通量测序和生物信息学手段,分析研究了两种相对封闭环境的微生物群落特征,发现了一些鲜有报道但有重要作用的菌种。肺炎病患下呼吸道宏基因组学研究中,为避免上呼吸道及口腔菌群的干扰,选取病人的支气管肺泡灌洗液为样本,获得了下呼吸道样品总DNA。通过反向杂交筛选方法,利用链霉亲和素磁珠偶联生物素探针,杂交去除人类基因组DNA。经过进一步Real-time PCR检测和454焦磷酸测序,证明经过处理的样本不仅保持了高丰度菌群的群落特征,还对中低丰度菌群有良好的覆盖。通过生物信息学分析,鉴定了20种呼吸道重症感染相关细菌物种,发现了两种呼吸道潜在致病菌Microbacterium laevaniformans和Borrelia garinii,检测到4种耐药基因和6种细菌侵染所需的毒力因子。宏基因组学研究结果与患者的临床症状相吻合,为快速诊断及合理用药提供了依据。沼气发酵体系宏基因组学研究中,比较并优化了总DNA的提取方法。利用454焦磷酸测序技术和生物信息分析手段,获得了厌氧发酵产甲烷体系中微生物群落的整体特征,并发现不同提取方法中细胞裂解手段与裂解程度对后续菌群分析有着显著影响。对菌群分布的研究发现,高丰度菌群主要参与蛋白质、脂肪、纤维素以及其他大分子聚合物降解。通过基因功能注释检测到体系内存在食乙酸和食氢两种代谢途径。本体系中新发现两种优势菌属Psychrobacter和Anaerococcus。其中,Psychrobacter含有低温脂肪水解酶,在中低温发酵应用中具有潜在价值。而Anaerococcus属在本研究体系中大量存在,也揭示它在生物质降解产甲烷过程中的巨大潜能。这些研究结果,为优化中低温固态厌氧沼气发酵工艺、提高甲烷产气量提供了依据。
[Abstract]:As an emerging field of research, macrogenomics has opened up new space for microbial research. With its high and new technology, macrogenomics has penetrated into many fields of complex environmental research, such as oceans, soils, hot springs, etc. Human oral and gastrointestinal tract, alternative energy, environmental repair, agriculture and biological defense. Normal human lower respiratory tract is a relatively closed sterile environment, Bacteria infecting the lower respiratory tract will pose a great threat to human health and even life. At present, the study of microflora in human lower respiratory tract is limited by sampling methods and other problems. But in other closed environments outside the human environment, such as biogas fermentation system, the study of macrogenomics is still in its infancy. It can reveal the functional characteristics of microflora in fermentation system, which is of great significance to improve methane gas production and guide industrial production. In this paper, the lower respiratory tract and biogas fermentation system of pneumonia patients were selected as the representatives of human body and external environment. The methods of obtaining DNA from different samples were optimized, and the methods of high throughput sequencing and bioinformatics were used. The characteristics of microbe community in two relatively closed environments were analyzed and some species which were rarely reported but played an important role were found. In order to avoid the interference of upper respiratory tract and oral microflora in the study of lower respiratory tract macrogenomics in patients with pneumonia, the bronchoalveolar lavage fluid was selected as a sample, and the total DNA of lower respiratory tract samples was obtained. The human genome DNAA was removed by hybridization with streptavidin magnetic beads coupled biotin probe. After further Real-time PCR detection and 454 pyrosequencing, it was proved that the treated samples not only maintained the community characteristics of high abundance bacteria, Through bioinformatics analysis, 20 species of bacteria associated with severe respiratory tract infection were identified. Two potential respiratory pathogens, Microbacterium laevaniformans and Borrelia Garinii, were found, and four drug-resistant genes and six virulence factors for bacterial infection were detected. The results of macrogenomics were consistent with the clinical symptoms of the patients. It provides a basis for rapid diagnosis and rational use of drugs. In the study of macrogenomics of biogas fermentation system, the extraction methods of total DNA were compared and optimized. By means of 454 pyrosequencing and bioinformatics analysis, the overall characteristics of microbial community in anaerobic fermentation methanogenic system were obtained. It was found that the cell cleavage and the degree of cleavage had a significant effect on the subsequent microflora analysis in different extraction methods. It was found that the high abundance microflora was mainly involved in protein and fat. Degradation of cellulose and other macromolecular polymers. Two metabolic pathways, acetic acid and hydrogen, were detected by gene functional annotation. Two dominant bacteria, Psychrobacter and Anaerococcus, were found in this system. Psychrobacter contains low-temperature fatty hydrolase. It has potential value in the application of medium and low temperature fermentation. However, Anaerococcus is widely present in this research system, which also reveals its great potential in the process of biomass degradation and methane production. These results are the optimization of medium and low temperature solid anaerobic biogas fermentation process. Increasing methane gas production is the basis.
【学位授予单位】：中国科学院北京基因组研究所
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.1;Q78

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