本文關(guān)鍵詞：RNA高通量測序技術(shù)以及魷魚—費氏弧菌共生機(jī)制的研究　出處：《東南大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 新一代高通量測序 測序文庫制備 RNA 共生 微生物 殼質(zhì)(糖)

【摘要】：高通量測序技術(shù)推動了生命科學(xué)領(lǐng)域研究的快速發(fā)展。新一代測序技術(shù)在靈敏度、精確性、廉價和通量等方面都比傳統(tǒng)的基于Sanger的測序技術(shù)具有很大的優(yōu)勢,是當(dāng)前應(yīng)用最廣泛的測序技術(shù),目前已應(yīng)用于腫瘤學(xué)、遺傳學(xué)、免疫學(xué)、病原學(xué)、微生物學(xué)、藥學(xué)、細(xì)菌分類學(xué)、比較基因組學(xué)和演化基因組學(xué)以及轉(zhuǎn)錄組學(xué)等多個領(lǐng)域。RNA-Seq,即轉(zhuǎn)錄組測序,是新一代測序技術(shù)的主要應(yīng)用之一,可以對生物樣本不同發(fā)育狀態(tài)的基因表達(dá)進(jìn)行全面監(jiān)測。RNA-Seq技術(shù)相比微陣列技術(shù),可對無參考基因組序列的全基因組cDNA進(jìn)行高通量測序。高通量測序樣本制備是測序研究中的第一步,也是最關(guān)鍵的步驟之一。目前,各大高通量測序平臺中樣本制備通常存在步驟繁瑣、并行性差、價格昂貴等問題,且對原始樣本的輸入量要求高,因此測序文庫制備方案的優(yōu)化一直是高通量測序技術(shù)的研究熱點。共生是兩種或更多的物種個體之間的密切長期的關(guān)系。共生物之間的代謝協(xié)作是互益的共生關(guān)系進(jìn)化的核心要素,尤其是宿主或者共生物提供的營養(yǎng)可以驅(qū)動共生的共進(jìn)化,共發(fā)展以及生態(tài)系統(tǒng)的搭建。近年來,研究者應(yīng)用高通量測序手段探索哺乳動物腸道微生物的基因表達(dá)以及免疫反應(yīng)的晝夜節(jié)律特性。這些節(jié)律可以影響腸道微生物群,以及它們在正常組織發(fā)育、營養(yǎng)代謝以及疾病防御方面的作用；然而,關(guān)于微生物基因表達(dá)對宿主源營養(yǎng)的反饋的研究甚少。本論文從方法學(xué)研究入手,在“質(zhì)”和“量”兩個方面優(yōu)化了高通量測序平臺的文庫制備方法。在此基礎(chǔ)上,基于夏威夷短尾魷魚-費氏弧菌雙生物共生模型,分析了費氏弧菌ES114菌種在不同的培養(yǎng)環(huán)境中的轉(zhuǎn)錄組表達(dá)差異,探索了共生的宿主晝夜節(jié)律地提供的殼質(zhì)對費氏弧菌基因表達(dá)及其代謝網(wǎng)絡(luò)的調(diào)控規(guī)律,從生理學(xué)和化學(xué)層面揭示了宿主晝夜節(jié)律地提供的營養(yǎng)的生物學(xué)意義。本論文主要內(nèi)容如下：一、高通量測序文庫制備方法的優(yōu)化本論文對高通量測序平臺的miRNA文庫制備方法進(jìn)行了優(yōu)化,將一對兩端修飾的單鏈RNA接頭替換原有的雙鏈接頭,分兩步加入反應(yīng)體系,使RNA接頭只能特異性地與目標(biāo)序列的3’端或5’端連接。改進(jìn)的制備方案減少了71%的雙鏈接頭自連接序列,有效地提高了測序通量。本論文還提出了一種可進(jìn)行單分子核酸擴(kuò)增的磁珠模型,并將其應(yīng)用于核酸測序文庫的制備中。通過優(yōu)化磁珠表面的化學(xué)修飾以及固相載體對引物的連接,提高了磁珠表面核酸的擴(kuò)增效率,可實現(xiàn)待測序的核酸序列在磁珠表面的高效連接和擴(kuò)增,擴(kuò)增后的磁珠表面包含單拷貝的待測核酸序列。該測序文庫制備方法具有操作簡便、文庫制備效率高、成本低、對初始樣本量要求低等優(yōu)勢,可應(yīng)用于多種高通量測序平臺的文庫制備。二、優(yōu)化的文庫制備方法在孕婦血漿游離miRNA分析中的研究本論文應(yīng)用改進(jìn)的文庫制備方法對來自23位孕婦外周血中的超過300個游離niRNA進(jìn)行了測序分析,優(yōu)化的文庫制備方案維持了較高的保真度,制備出理想的niRNA測序文庫。論文挑選了孕婦血漿中的特異性miRNA,測序結(jié)果分析顯示19例孕婦外周血游離miRNA在產(chǎn)后表達(dá)下調(diào)超過兩倍。熒光定量RT-PCR技術(shù)驗證了高通量測序技術(shù)在miRNA表達(dá)分析中的準(zhǔn)確性。研究還發(fā)現(xiàn)子癇患者血漿三個特異性游離miRNA的表達(dá)均超出正常孕婦兩倍以上,為高通量測序技術(shù)在妊高癥等疾病的無創(chuàng)性產(chǎn)前診斷奠定了基礎(chǔ)。三、高通量測序技術(shù)在魷魚-費氏弧菌共生系統(tǒng)細(xì)菌轉(zhuǎn)錄組表達(dá)分析中的應(yīng)用殼質(zhì)是一種普遍存在于動植物共生關(guān)系中,來源于宿主的營養(yǎng)和信號。本論文應(yīng)用高通量測序平臺,對費氏弧菌在不同培養(yǎng)環(huán)境中的轉(zhuǎn)錄組表達(dá)差異進(jìn)行了分析,發(fā)現(xiàn)了殼質(zhì)顯著影響了細(xì)菌的殼質(zhì)運輸和代謝通路相關(guān)基因的表達(dá)。RNA-Seq結(jié)果顯示,當(dāng)費氏弧菌在含有殼質(zhì)糖的培養(yǎng)液中生長時,運輸殼質(zhì)的PTS通路的nagE, nagE2, crr;糖酵解通路中g(shù)lpF/k；甚至是呼吸通路中的ccoN, sdhC等超過30個基因呈現(xiàn)出兩倍以上的表達(dá)差異。該結(jié)論為進(jìn)一步分析共生系統(tǒng)宿主發(fā)育過程中內(nèi)環(huán)境的化學(xué)和生理學(xué)動態(tài)發(fā)展提供了新的線索,也為研究復(fù)雜的共生系統(tǒng)甚至哺乳動物中宿主源的營養(yǎng)對代謝調(diào)控的保守機(jī)制提供了途徑。四、魷魚-費氏弧菌共生系統(tǒng)的化學(xué)對話和生理學(xué)發(fā)展基于RNA-Seq的分析結(jié)果,本論文針對具有發(fā)光器官的夏威夷短尾魷魚和發(fā)冷光的細(xì)菌費氏弧菌這個雙生物共生模型,借助細(xì)菌克隆、熒光免疫細(xì)胞化學(xué)、共聚焦顯微鏡、細(xì)菌耐酸反饋指示等研究工具,發(fā)現(xiàn)成熟宿主的血細(xì)胞節(jié)律性地將殼質(zhì)釋放到發(fā)光器官腺窩內(nèi),導(dǎo)致腺窩內(nèi)節(jié)律性的酸化；該營養(yǎng)對弧菌代謝通路有三個主要調(diào)控作用：1)共生菌通過PTS運輸通路優(yōu)先運輸殼質(zhì)進(jìn)入弧菌內(nèi),2)代謝殼質(zhì)抑制醋酸鹽代謝,造成共生內(nèi)環(huán)境的酸化,3)降低呼吸通路對氧氣的消耗,進(jìn)而支持共生菌夜間發(fā)光,滿足宿主的生理學(xué)需求。該研究揭示了殼質(zhì)這種宿主源的營養(yǎng)如何從生理學(xué)和化學(xué)層面調(diào)控共生物代謝,解釋了宿主晝夜節(jié)律性地提供這種營養(yǎng)的生物學(xué)意義。上述研究驗證了微生物測序結(jié)果的準(zhǔn)確性,高通量測序技術(shù)在共生系統(tǒng)微生物的轉(zhuǎn)錄組表達(dá)差異分析中的研究是揭示共生系統(tǒng)中宿主源的營養(yǎng)對共生菌的代謝調(diào)控的有效方法。魷魚-費氏弧菌共生模型為研究宿主和共生菌的化學(xué)交互提供了基礎(chǔ),它可以為縱向研究節(jié)律關(guān)聯(lián)的發(fā)展提供簡單有效的方法,不僅對發(fā)展生物學(xué),細(xì)菌學(xué)和時間生物學(xué)等更廣的領(lǐng)域具有重大意義,對于我們探索微生物對人類健康的影響中的保守主題也提供了重要價值和途徑。
[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.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q78

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