A Study on Transcriptomic Profiling of Differentially Expressed Genes Relevant to Innate and Adaptive Immune Pathways in Transge

University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 5-24-2018 A Study on Transcriptomic Profiling of Differentially Expressed Genes Relevant to Innate and Adaptive Immune Pathways in Transgenic Rainbow Trout (Oncorhynchus mykiss) Harboring Cecropin P1 Transgene Yueh-Chiang Han University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Han, Yueh-Chiang, "A Study on Transcriptomic Profiling of Differentially Expressed Genes Relevant to Innate and Adaptive Immune Pathways in Transgenic Rainbow Trout (Oncorhynchus mykiss) Harboring Cecropin P1 Transgene" (2018). Doctoral Dissertations. 1841. https://opencommons.uconn.edu/dissertations/1841 A Study on Transcriptomic Profiling of Differentially Expressed Genes Relevant to Innate and Adaptive Immune Pathways in Transgenic Rainbow Trout (Oncorhynchus mykiss) Harboring Cecropin P1 Transgene Yueh-Chiang Han, PhD University of Connecticut, 2018 Abstract Introduction of antimicrobial peptide (AMP) genes into fish genome by the transgenic technology provided a promising solution to control fish disease. Cecropin P1, a known porcine origi- nated AMP, had been introduced into the rainbow trout genome for production of transgenic fish by Chiou et al. (2014) in Chen’s laboratory. Repeated, in vivo, challenge studies demonstrated that these transgenic fish exhibited resistant characteristic to infection by microbial pathogens. Here, we hypothesized that cecropin P1 transgene product may not only directly eliminate microbial pathogens, but also indirectly exert immunomodulatory activity in the transgenic hosts to elevate their disease resistance. To address this hypothesis, I have employed the technolo- gies of mRNA deep sequencing (mRNA-Seq) and reverse transcription quantitative real-time PCR (RT-qPCR) array. In a preliminary cDNA microarray analysis, Lo et al. (2014) reported functional perturbations of the immune relevant pathways in three immune competent tissues (namely, the spleen, kidney and liver) of the cecropin P1 transgenic rainbow trout. To overcome the technical limitations of the cDNA microarray, the differentially expressed genes (DEGs) of immune relevant pathways in the spleen, kidney and liver of two families of transgenic rainbow trout was analyzed by de novo mRNA-Seq. By sorting of Reads Per Kilobase of transcript per million mapped reads, the DEGs were determined. From the GeneCodis enrichment analysis, functional alterations of biological processes with identifiers of Gene Ontology: Biological Pro- cess and Kyoto Encyclopedia of Genes and Genomes databases were observed. Via pathway analyses, immune relevant processes in the spleen and kidney, and energy metabolism relevant processes in the liver were significantly perturbed. By employing a custom-layout RT-qPCR array with pre-defined immune relevant DEGs, the expression patterns in three remaining transgenic families were profiled. The results of mRNA-Seq and RT-qPCR array analyses supported the hypothesis that the combined effects of transgenic cecropin P1 elevate the host disease resistance. Furthermore, the RT-qPCR array with pre-defined DEGs as bio-markers promises a fast, effective and inexpensive technique for screening of disease resistant fish strains for aqua- culture. A Study of Transcriptomic Profiling of Differentially Expressed Genes Relevant to Innate and Adaptive Immune Pathways in Transgenic Rainbow Trout (Oncorhynchus mykiss) Harboring Cecropin P1 Transgene Yueh-Chiang Han B.S., National Chung-Hsing University, 2000 M.S., National Taiwan University, 2002 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2018 APPROVAL PAGE Doctor of Philosophy Dissertation A Study of Transcriptomic Profiling of Differentially Expressed Genes Relevant to Innate and Adaptive Immune Pathways in Transgenic Rainbow Trout (Oncorhynchus mykiss) Harboring Cecropin P1 Transgene Presented by Yueh-Chiang Han, B.S., M.S. Major Advisor: Thomas T. Chen, Ph.D Associate Advisor: Johann Peter Gogarten, Ph.D Associate Advisor: Joerg Graf, Ph.D Associate Advisor: Charles Giardina, Ph.D Associate Advisor: Ping Zhang, Ph.D University of Connecticut 2018 ii Acknowledgements First and foremost, I will be eternally grateful for my advisor, Dr. Thomas Chen. Six years ago, he provided me with an opportunity to pursue research, and has never given up on me. His guidance throughout my graduate work is something I will always be thankful for. Dr. Chen and his team members have equipped me with invaluable skills for a successful journey into my scientific career. I was taught how to think critically, how to connect with others, and how to tell a story with science that can leave a lasting impact. I would like to thank my doctoral advisory committee, Dr. Peter Gogarten, Dr. Joerg Graf, Dr. Charles Giardina, and Dr. Ping Zhang for their generosity in providing their time and thought- ful insights into my research. Each one of them has been uniquely influential on my graduate career and I appreciate them all so much. The Chen’s lab members, including Dr. CM Lin, Dr. Jay Lo, Dr. Jimmy Yeh and Maria Chen et al., should have my gratitude as well. Activities including lots of discussion about the researches, generating exciting topic, solving difficult problems encountered will not be forgot- ten. Additionally, I greatly appreciated Dr. Dong-Guk Shin and the team members, e.g. Tham Hoang, Pujan Joshi, Yue Zhao, Stephanie Piekos and Cameron Reilly et al., for an opportunity of independent study about pathway editor development and bioinformatics training. This independent study greatly expands my academic ability and stimulate my research interests to cancer immune-oncology area. My experiences in teaching assistant was valuable to me as well. As an international student, to teach in second language is considered the most challenge in my entire life. Without assistance from supervisors and colleagues in Biol1107 and MCB2000, I cannot survive these real challenges. Because of them, I improved my ability in communication and had fun in teaching in the past few years. I also want to present my appreciation to the faculties and staff at the MCB department, and all friendly students in this community, for financial support and all kind of assistance. Last but not least, I would like to thank my two families, my original family and spiritual family. Firstly, my parents, my sister and other family members, knowing they will always support my back, allows me to focus on pursuing my research to achieve academic goals. Lastly, the student organization of International Christian Fellowship is considered as my spiritual family. The support and inspiration from ICF members greatly motivate my inner strength so that I can overcome any encountered challenges. iii Contents Approval Page ii Acknowledgements iii List of Figures vii Abbreviations ix 1 General Introduction and Background1 1.1 Overview of Anti-microbial Peptides.......................1 1.2 The Structures of Cecropins and Their Interaction with the Plasma Membrane of Microbial Pathogens..............................4 1.3 The Regulation of Cecropins...........................9 1.4 Immunomodulatory Activities of AMPs Serving as Indirect Effects....... 11 1.5 Overview of Transgenic Fish Technology.................... 17 1.6 Powerful Tools of Genomic Approaches to Profile the Expression of Immune Relevant Genes in Transgenic Fish........................ 25 1.7 Research Rationale and Objectives........................ 33 2 General Materials and Methods 36 2.1 Nucleic Acid Isolation and Extraction...................... 36 2.1.1 RNA Extraction (guanidium isothiocyanate)............... 36 2.1.2 RNA Extraction (TRIzol)......................... 38 2.1.3 Genomic DNA Removal via DNase Digestion.............. 38 2.2 Reverse Transcription Synthesis of First Strand cDNA............. 39 2.3 Polymerase Chain Reaction (PCR)........................ 40 2.4 Quantitative Real-Time PCR (RT-qPCR)..................... 40 3 RNA-Seq Analysis of Differentially Expressed Genes Relevant to Innate and Adap- tive Immunity in Cecropin P1 Transgenic Rainbow Trout (Oncorhynchus mykiss) 42 3.1 Introduction.................................... 42 3.2 Special Materials and Methods.......................... 46 iv 3.2.1 Transcriptomic Sequencing and Reads Assembly............ 46 3.2.2 Unigene Annotation and Functional Classification........... 47 3.2.3 Differential Expression of Unigenes................... 47 3.2.4 Bioinformatics Analysis......................... 48 3.3 Results....................................... 48 3.3.1 De novo Sequencing, Reads Assembly, and Annotation of Reference Database from Non-transgenic Fish................... 48 3.3.2 Sequencing and Assembly of Cecropin P1 Transgenic Rainbow Trout. 51 3.3.3 Determination of Differential Expression Profiles............ 54 3.3.4 Enrichment analysis of biology terms of GO and KEGG pathways by GeneCodis................................ 58 3.3.5 Analysis of Functional Perturbed Immune Relevant Pathways..... 62 3.4 Discussion..................................... 65 3.4.1 Spleen: the Major Innate/Adaptive Immune Relevant Organ...... 65 3.4.2 Kidney: the Major Hematopoiesis Relevant Organ........... 69 3.4.3 Liver: the Major Energy Efficiency Relevant Organ........... 71 3.5 Author’s Contribution of RNA-Seq Analysis................... 73 4 An Immune Relevant

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