RNA高通量测序技术以及鱿鱼—费氏弧菌共生机制的研究

发布时间：2018-01-15 17:22

本文关键词：RNA高通量测序技术以及鱿鱼—费氏弧菌共生机制的研究　出处：《东南大学》2016年博士论文　论文类型：学位论文

【摘要】：高通量测序技术推动了生命科学领域研究的快速发展。新一代测序技术在灵敏度、精确性、廉价和通量等方面都比传统的基于Sanger的测序技术具有很大的优势,是当前应用最广泛的测序技术,目前已应用于肿瘤学、遗传学、免疫学、病原学、微生物学、药学、细菌分类学、比较基因组学和演化基因组学以及转录组学等多个领域。RNA-Seq,即转录组测序,是新一代测序技术的主要应用之一,可以对生物样本不同发育状态的基因表达进行全面监测。RNA-Seq技术相比微阵列技术,可对无参考基因组序列的全基因组cDNA进行高通量测序。高通量测序样本制备是测序研究中的第一步,也是最关键的步骤之一。目前,各大高通量测序平台中样本制备通常存在步骤繁琐、并行性差、价格昂贵等问题,且对原始样本的输入量要求高,因此测序文库制备方案的优化一直是高通量测序技术的研究热点。共生是两种或更多的物种个体之间的密切长期的关系。共生物之间的代谢协作是互益的共生关系进化的核心要素,尤其是宿主或者共生物提供的营养可以驱动共生的共进化,共发展以及生态系统的搭建。近年来,研究者应用高通量测序手段探索哺乳动物肠道微生物的基因表达以及免疫反应的昼夜节律特性。这些节律可以影响肠道微生物群,以及它们在正常组织发育、营养代谢以及疾病防御方面的作用；然而,关于微生物基因表达对宿主源营养的反馈的研究甚少。本论文从方法学研究入手,在“质”和“量”两个方面优化了高通量测序平台的文库制备方法。在此基础上,基于夏威夷短尾鱿鱼-费氏弧菌双生物共生模型,分析了费氏弧菌ES114菌种在不同的培养环境中的转录组表达差异,探索了共生的宿主昼夜节律地提供的壳质对费氏弧菌基因表达及其代谢网络的调控规律,从生理学和化学层面揭示了宿主昼夜节律地提供的营养的生物学意义。本论文主要内容如下：一、高通量测序文库制备方法的优化本论文对高通量测序平台的miRNA文库制备方法进行了优化,将一对两端修饰的单链RNA接头替换原有的双链接头,分两步加入反应体系,使RNA接头只能特异性地与目标序列的3’端或5’端连接。改进的制备方案减少了71%的双链接头自连接序列,有效地提高了测序通量。本论文还提出了一种可进行单分子核酸扩增的磁珠模型,并将其应用于核酸测序文库的制备中。通过优化磁珠表面的化学修饰以及固相载体对引物的连接,提高了磁珠表面核酸的扩增效率,可实现待测序的核酸序列在磁珠表面的高效连接和扩增,扩增后的磁珠表面包含单拷贝的待测核酸序列。该测序文库制备方法具有操作简便、文库制备效率高、成本低、对初始样本量要求低等优势,可应用于多种高通量测序平台的文库制备。二、优化的文库制备方法在孕妇血浆游离miRNA分析中的研究本论文应用改进的文库制备方法对来自23位孕妇外周血中的超过300个游离niRNA进行了测序分析,优化的文库制备方案维持了较高的保真度,制备出理想的niRNA测序文库。论文挑选了孕妇血浆中的特异性miRNA,测序结果分析显示19例孕妇外周血游离miRNA在产后表达下调超过两倍。荧光定量RT-PCR技术验证了高通量测序技术在miRNA表达分析中的准确性。研究还发现子痫患者血浆三个特异性游离miRNA的表达均超出正常孕妇两倍以上,为高通量测序技术在妊高症等疾病的无创性产前诊断奠定了基础。三、高通量测序技术在鱿鱼-费氏弧菌共生系统细菌转录组表达分析中的应用壳质是一种普遍存在于动植物共生关系中,来源于宿主的营养和信号。本论文应用高通量测序平台,对费氏弧菌在不同培养环境中的转录组表达差异进行了分析,发现了壳质显著影响了细菌的壳质运输和代谢通路相关基因的表达。RNA-Seq结果显示,当费氏弧菌在含有壳质糖的培养液中生长时,运输壳质的PTS通路的nagE, nagE2, crr;糖酵解通路中glpF/k；甚至是呼吸通路中的ccoN, sdhC等超过30个基因呈现出两倍以上的表达差异。该结论为进一步分析共生系统宿主发育过程中内环境的化学和生理学动态发展提供了新的线索,也为研究复杂的共生系统甚至哺乳动物中宿主源的营养对代谢调控的保守机制提供了途径。四、鱿鱼-费氏弧菌共生系统的化学对话和生理学发展基于RNA-Seq的分析结果,本论文针对具有发光器官的夏威夷短尾鱿鱼和发冷光的细菌费氏弧菌这个双生物共生模型,借助细菌克隆、荧光免疫细胞化学、共聚焦显微镜、细菌耐酸反馈指示等研究工具,发现成熟宿主的血细胞节律性地将壳质释放到发光器官腺窝内,导致腺窝内节律性的酸化；该营养对弧菌代谢通路有三个主要调控作用：1)共生菌通过PTS运输通路优先运输壳质进入弧菌内,2)代谢壳质抑制醋酸盐代谢,造成共生内环境的酸化,3)降低呼吸通路对氧气的消耗,进而支持共生菌夜间发光,满足宿主的生理学需求。该研究揭示了壳质这种宿主源的营养如何从生理学和化学层面调控共生物代谢,解释了宿主昼夜节律性地提供这种营养的生物学意义。上述研究验证了微生物测序结果的准确性,高通量测序技术在共生系统微生物的转录组表达差异分析中的研究是揭示共生系统中宿主源的营养对共生菌的代谢调控的有效方法。鱿鱼-费氏弧菌共生模型为研究宿主和共生菌的化学交互提供了基础,它可以为纵向研究节律关联的发展提供简单有效的方法,不仅对发展生物学,细菌学和时间生物学等更广的领域具有重大意义,对于我们探索微生物对人类健康的影响中的保守主题也提供了重要价值和途径。
[Abstract]:High throughput sequencing technology to promote the rapid development of the life science research field. The accuracy of a new generation of sequencing technology in sensitivity, cheap and flux etc. compared with the traditional Sanger based sequencing technology has a great advantage, is currently the most widely used sequencing technology, at present has been used in oncology, immunology, genetics, the etiology, microbiology, pharmacy, bacterial taxonomy, comparative genomics and.RNA-Seq multiple field of evolutionary genomics and transcriptomics, namely transcriptome sequencing, is one of the main applications of a new generation of sequencing technology, biological samples can be expressed on different developmental status of comprehensive monitoring technology.RNA-Seq gene microarray technology can be compared. High throughput sequencing of the whole genome of cDNA reference genome sequences. The high-throughput sequencing sample preparation is the first step in the study of sequencing, is one of the most critical steps At present, the high-throughput sequencing platform in sample preparation is usually tedious, parallelism, and expensive, and the input of the original sample requirements, so the optimization of preparation of sequencing library system has been a research hotspot in high-throughput sequencing technology. Symbiosis is between two or more species individual close long-term relationship. Coordination between the metabolism of biological evolution is the core elements of the symbiotic relationship of mutual benefit, especially the co evolution of host or common biological provided nutrition can drive symbiosis, build common development and ecosystem. In recent years, the circadian rhythm characteristics of researchers used high-throughput sequencing methods to explore gene expression the mammalian intestinal microflora and immune response. These rhythms can influence the gut microbes, and they are in normal tissue development, nutrient metabolism and disease defense. However, a little; to study the feedback source nutrition of host microbial gene expression. This paper studies the method of the two aspects of "quality" and "quantity" to optimize the library preparation method of high-throughput sequencing platform. On this basis, Hawaii bobtail squid - Vibrio fischeri double biological symbiosis model based on the analysis of the Vibrio fischeri ES114 strains in different culture conditions in the transcriptome differences, explore the regulation of symbiotic host provide circadian rhythm of the shell of Vibrio fischeri gene expression and metabolic network, reveals the circadian rhythm of the host to provide nutrition and biological significance from the physiological and chemical aspects. The main contents this thesis is as follows: first, the optimization of high-throughput sequencing library preparation method of the high-throughput sequencing platform miRNA library preparation method was optimized, a modification to the end Single stranded RNA joint replacement double chain joint of the original two steps into the reaction system, so that the RNA joint only specifically with the target sequence in 3 'or 5' end connection. Preparation scheme improved to reduce the double chain joint 71% self connected sequence, effectively improve the sequencing throughput. This thesis also put forward a model of magnetic single molecular nucleic acid amplification, and its application in nucleic acid sequencing library preparation. By optimizing the bead surface chemical modification and solid support primer connection, improve the amplification efficiency of the bead surface of nucleic acid, can realize the target DNA sequence on the bead surface efficient connection the surface of the magnetic beads and amplified, amplified contains a single copy of the target DNA. The sequencing library preparation method has the advantages of simple operation, high efficiency of library preparation, low cost, low requirement on the advantages of the initial sample volume, can be applied to a variety of high Library preparation of high-throughput sequencing platform. On the two, 23 pregnant women from the peripheral blood of pregnant women in the library preparation method of plasma free miRNA in the analysis of the optimization of library preparation method to improve the application of more than 300 free niRNA were sequenced and the scheme of library preparation optimization to maintain a high fidelity, prepared by niRNA sequencing library. The ideal selection of specific miRNA in maternal plasma, the sequencing analysis showed that the expression of peripheral blood lymphocytes in 19 pregnant women in the postpartum free miRNA down more than two times. The fluorescence quantitative RT-PCR technology to verify the high-throughput sequencing technology in miRNA expression accuracy analysis. The study also found that the expression of three patients with eclampsia specific plasma free miRNA were all higher than the normal pregnant women more than two times, for high-throughput sequencing technology in pregnancy induced hypertension and other diseases of the non-invasive prenatal diagnosis of the foundation. Three, high throughput Sequencing bacterial symbiotic system transcriptome in squid - Vibrio fischeri expression analysis in the application of chitin is a ubiquitous in plants and symbiotic relationship, from the host nutrition and signal. The application of high-throughput sequencing platform of Vibrio fischeri in different culture conditions in the transcriptome differences were analyzed, found the shell has significant influence on the expression of.RNA-Seq results in chitin transport and metabolism pathway genes in bacteria showed that when Vibrio fischeri in liquid culture containing chitin sugar when grown in PTS transport pathway chitinous nagE, nagE2, CRR; the glycolytic pathway of glpF/k; even the respiratory pathway in ccoN, more than sdhC 30 genes showing differential expression of more than two times. The results provide useful clues for further analysis of dynamic development of chemical and biological symbiosis system in the development process of the internal environment of the host, but also Provides a way of conservative mechanism symbiotic system for the study of complex even in mammals host source nutrition on metabolic regulation. Four, squid - Vibrio fischeri symbiotic chemical dialogue system and physiological development based on the results of RNA-Seq analysis, this paper has a luminous organ Hawaii bobtail squid and luminescent bacteria Vibrio fischeri this double biological symbiosis model, with the aid of bacterial cloning, immunofluorescence, confocal microscopy, acid feedback indication and other research tools, found the rhythm of mature blood cells of host will be released to the light organ chitinous crypt, gland fossa leads to acidification of rhythm; the nutrition has three main effects on Vibrio metabolic pathways: 1) symbiotic bacteria transport through the PTS pathway in priority transport into 2) shell Vibrio, metabolic inhibition of acetate metabolism caused by chitin, symbiotic environment acid, fall 3) Low consumption of respiratory pathway on oxygen, and thus support the symbiotic bacteria of light at night, meet the host physiology demand. The study reveals the source of nutrition to the host shell from the physiological and chemical regulation level total biological metabolism, explain the host circadian rhythm to provide nutrition. The biological significance of this study to verify the accuracy of microorganisms the sequencing results, high-throughput sequencing technology in the symbiotic system of microbial transcriptome study on differential expression analysis is the effective way to reveal the source of symbiotic host nutrition on metabolic regulation of symbiotic bacteria. Squid - Vibrio fischeri symbiosis model interaction is provided for the study of host and symbiotic bacteria chemical basis, provides a simple and effective method the development of it can be for a longitudinal study of rhythm related, not only to the development of biology, bacteriology and biology of the broader field of time is of great significance It also provides important values and ways for us to explore the conservative themes of the impact of microbes on human health.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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