本文選題：宏基因組學(xué)　切入點(diǎn)：焦磷酸測(cè)序　出處：《中國(guó)科學(xué)院北京基因組研究所》2013年博士論文

【摘要】：作為一個(gè)新興的研究領(lǐng)域,宏基因組學(xué)為微生物研究開(kāi)拓了新的空間。宏基因組學(xué)憑借其高新的技術(shù),已經(jīng)滲透到眾多復(fù)雜環(huán)境研究領(lǐng)域,例如海洋、土壤、熱泉、人體口腔及胃腸道、替代能源、環(huán)境修復(fù)、農(nóng)業(yè)和生物防御等。正常人體下呼吸道是一個(gè)相對(duì)封閉的無(wú)菌環(huán)境,細(xì)菌侵染下呼吸道會(huì)對(duì)人體健康乃至生命造成極大的威脅。目前人體下呼吸道微生物組研究由于受到取樣方法等問(wèn)題的限制,研究尚不夠深入。而在人體環(huán)境之外其他封閉環(huán)境中,例如沼氣發(fā)酵體系,宏基因組學(xué)研究也尚在起步階段。沼氣發(fā)酵體系宏基因組學(xué)研究,可以揭示發(fā)酵體系內(nèi)菌群功能特征,對(duì)提高甲烷產(chǎn)氣量、指導(dǎo)工業(yè)化生產(chǎn)有重要意義。 本文針對(duì)人體內(nèi)、外環(huán)境分別選取肺炎病患下呼吸道和沼氣發(fā)酵體系為代表,優(yōu)化了不同樣品DNA獲得方法,通過(guò)高通量測(cè)序和生物信息學(xué)手段,分析研究了兩種相對(duì)封閉環(huán)境的微生物群落特征,發(fā)現(xiàn)了一些鮮有報(bào)道但有重要作用的菌種。 肺炎病患下呼吸道宏基因組學(xué)研究中,為避免上呼吸道及口腔菌群的干擾,選取病人的支氣管肺泡灌洗液為樣本,獲得了下呼吸道樣品總DNA。通過(guò)反向雜交篩選方法,利用鏈霉親和素磁珠偶聯(lián)生物素探針,雜交去除人類基因組DNA。經(jīng)過(guò)進(jìn)一步Real-time PCR檢測(cè)和454焦磷酸測(cè)序,證明經(jīng)過(guò)處理的樣本不僅保持了高豐度菌群的群落特征,還對(duì)中低豐度菌群有良好的覆蓋。通過(guò)生物信息學(xué)分析,鑒定了20種呼吸道重癥感染相關(guān)細(xì)菌物種,發(fā)現(xiàn)了兩種呼吸道潛在致病菌Microbacterium laevaniformans和Borrelia garinii,檢測(cè)到4種耐藥基因和6種細(xì)菌侵染所需的毒力因子。宏基因組學(xué)研究結(jié)果與患者的臨床癥狀相吻合,為快速診斷及合理用藥提供了依據(jù)。 沼氣發(fā)酵體系宏基因組學(xué)研究中,比較并優(yōu)化了總DNA的提取方法。利用454焦磷酸測(cè)序技術(shù)和生物信息分析手段,獲得了厭氧發(fā)酵產(chǎn)甲烷體系中微生物群落的整體特征,并發(fā)現(xiàn)不同提取方法中細(xì)胞裂解手段與裂解程度對(duì)后續(xù)菌群分析有著顯著影響。對(duì)菌群分布的研究發(fā)現(xiàn),高豐度菌群主要參與蛋白質(zhì)、脂肪、纖維素以及其他大分子聚合物降解。通過(guò)基因功能注釋檢測(cè)到體系內(nèi)存在食乙酸和食氫兩種代謝途徑。本體系中新發(fā)現(xiàn)兩種優(yōu)勢(shì)菌屬Psychrobacter和Anaerococcus。其中,Psychrobacter含有低溫脂肪水解酶,在中低溫發(fā)酵應(yīng)用中具有潛在價(jià)值。而Anaerococcus屬在本研究體系中大量存在,也揭示它在生物質(zhì)降解產(chǎn)甲烷過(guò)程中的巨大潛能。這些研究結(jié)果,為優(yōu)化中低溫固態(tài)厭氧沼氣發(fā)酵工藝、提高甲烷產(chǎn)氣量提供了依據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)院北京基因組研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R563.1;Q78

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 唐振華;朱義朗;;產(chǎn)AmpC酶和ESBLs陰溝腸桿菌的臨床分布及耐藥性分析[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2009年01期

2 閆曉淼;衛(wèi)皇f，

本文編號(hào)：1661296