RNA High-throughout Sequencing Technology And The Study Of Squid-V.fischeri Symbiosis Mechanism

High-throughput sequencing (HTS) technology promotes rapid development of research of life science. The next generation sequencing (NGS) has advantages in sensitivity, accuracy, cost and throughput over sequencing technology based on Sanger. NGS is the maturest sequencing technology with most widespread applications at present. It has widespread applications in disciplines like oncology, genetics, immunology, etiology, microbiology, pharmacy, germ taxonomy, comparative genomics and transcriptomics. RNA-Seq, transcriptome sequencing, is one of the key applications of NGS, through which we could monitor the overall picture of the gene expression of the biological samples in various development states. Rather than microarray techonology, RNA-Seq could sequence the whole genomic cDNA with no reference genomic sequence. HTS library preparation is the first and the most essential process in sequencing research. Currently, library preparation in major sequencing platforms usually have disadvantages like tedious procedures, poor parallelism and expensive cost, and require high input of samples. Therefore, the improvement of the library preparationhas has always been research focus of HTS research.Symbiosis is a close and long-term association among individuals of two or more species. Metabolic association among symbionts is the key factor in beneficial symbiosis evolution; specifically, host or symbiont-derived nutrient could promotethe co-evolution, co-development or the establishment of ecological system of symbiosis. Recently, HTS has been applied to the exploration of diel characteristics of gene expression and immune response of microbiota in mammal intestine. These characteristics affect intestinal microbiota and their roles in tissue development, nutrition metabolism and disease defense; however, how the host-derived nutrient affects the microbiota gene expression remains largely unknown.Methodology is the first focus, and HTS library preparation method is improved in quality and quantity in the thesis. Based on the research finding, the binary symbiosis model between squid Eurymna scolopes and its luminous bacterial partner, Vibrio fischeri, is used to analyze the transcriptomic difference of V.fischeri ESI 14 strain in different cultures and explores the regulation of chitin-rhythmically provided by host to V.fischeri gene expression and the metabolic network, and reveals the biological meaning of the diel provision of this nutrient by the host from the physiological and chemical perspective in this work.The thesis consists the following four parts:1. Optimization of the HTS library preparation methodThe HTS miRNA library preparation method is optimized in the thesis. The double-strand adaptor are replacedby a couple of single-strand RNA adaptor modified at both ends, which are added into the reaction system separately, and enables the RNA adaptors to execute ligation exclusively at 5’end or 3’end. The optimized method decreases the rate of self-ligation of the double-strand adaptors by 71%, and improves the sequencing throughput effectively.A magnetic bead for single nucleic acid amplification model for library preparation is also brought up in this work, which is applied in nucleic acid library preparation. The method improves the primer ligation and nucleic acid amplification efficiency on beads by optimizing the chemical modification and ligation of solid surface and primers. Copies of single target nucleic acid cover the beads after high-efficiency ligation and amplification. The method has advantages of easy manipulation, high efficiency, low cost and low requirement of sample amount of library preparation, which could be applied to library preparation in many HTS platforms.2. Sequencing circulating miRNAs in maternal plasma with optimized library preparation methodThe optimized library preparation method is applied to sequencingover 300 circulating miRNAs in plasma from 23 pregnant women in this thesis, which keeps high fidelity and prepares ideal miRNA library. The sequencing result of specific plasma miRNAs in prenatal plasma showsover 2-fold down-regulation of circulating miRNAs expressionafter delivery in 19 plasma samples. Fluorescent quantitative RT-PCR validates the accuracy of HTS in miRNA profiling analysis. The research also showsthe expression of three specificcirculating miRNAs in preeclampsia women plasma are higher than those in normal pregnant women by over 2-fold, which establishes basis for the use of HTS in noninvasive prenatal diagnosis such as preeclampsia.3. Application of NGS in analyzing bacterial transcriptomic profile in squid-V.fischeri symbiosisChitin, a host-derived nutrient and signal, universally exists in the symbiosis in most animals and plants. In this work, HTS is applied to the analyses of the transcriptome difference of V.fischeri in different cultures, and finds chitin significantly affect the expression of bacterial genes related to chitin transportation and metabolic pathways. RNA-Seq data indicates the expression change of over 30 genes, including nagE, nagE2, err in chitin transportation PTS pathway; glpF/k in glycolysis; and even ccoN, sdhC in respiration pathway are over 2-fold when cultivate V.fischeri in cultures with the presence of chitin. The result provides novel clue for the analyses of dynamic innate chemical environment and physiological development states in symbiosis, and provides a tool for studying the conservative regulation mechanism of metabolism by host-derived nutrient in complex symbiosis systems, even mammals.4. Chemical negotiation and physiological development in squid-V.fischeri symbiosisBased on RNA-Seq data, the thesis focuses on the binary symbiosis model between squid Eurymna scolopes and its luminous bacterial partner, Vibrio fischeri, finds the hemocytes rhythmically release chitin to the light organ crypts, and leads to rhythmic diel acidification in the crypt with tools of bacterial coloning, fluorescence immunoassay, confocal microscope and bacterial acid tolerance response (ATR). The nutrient has three main regulations in V.fischeri metabolic pathway:1) V.fischeri prioritizes chitin transportation by PTS,2) chitin catabolism inhibits acetate uptake, and acidifies the innate environment,3) chitin catabolism decreases oxygen consumption via respiration pathway, and thus supports the bioluminescence, which meets the nocturnal biological requirement of the host.The research reveals how the host-derived nutrient chitin regulates the symbiont catabolism from the physiological and chemical perspectives, and explains the biological meaning of the diel provision of the nutrient by the host.The research verifies the accuracy of the microbiota sequencing result, thereby shows the research of HTS in the analyses of symbionts differential transcriptomic expression is an effective method in revealing the regulation of the symbionts metabolism by host-derived nutrient.The squid-V.fischeri symbiosis model provides basis for the study of the chemical negotiation between host and symbiont, and a both simple and effective way to longitudinally study the development of the rhythmic association, which illuminate not only profound value in developmental biology, bacteriology and chronobiology, but also a key path in exploring the conservative roles microbiota play in human health.