Master’s Thesis 2017 60 ECTS The Department of Animal and Aquacultural Sciences (IHA) Characterization and expression analysis of the key genes for early development of swim bladder in Atlantic cod Yihang Wang Aquaculture I ACKNOWLEDGEMENT The work here presented was performed at Nofima ÅS, Norway, during 2016-2017. I would like to express my sincere gratitude to my supervisor, Øivind Andersen, for leading me into such an interesting and significant project. The experience of working with him was motive and fun. Thank you for all the constructive suggestion along the entire procedure of my work and constant encouragement during my study here. I am also thankful to Katrine Hånes Kirste, for her endless patience and kindness to help on my work in lab. Besides, I sincerely thank Ifrat Jahan Tamanna, Gerrit Timmerhaus, and Hanne Johnsen for all the answers of my questions. At last, I would like to thank Norwegian University of Life Sciences for offering me the opportunity to study and Nofima for supporting me to accomplish my experiments and thesis. Yihang Wang Ås, August 2017 II ABSTRACT Some genes have been proved critical for swim bladder inflation during early stages. Hence, our researches were focused on the investigation into the genetic features of the key genes to early development of Atlantic cod swim bladder. The elovl1, pbx1, psap, and sftpb genes were selected, and the expression modes during embryonic and larval stages were studied by qPCR quantification procedure. Genetic structures, multiple alignment, phylogeny of these genes were also investigated. Researches of Atlantic cod sftpb was further examined by studying relative expression levels in different organs and multiple genomic conserved synteny. Cod elovl1 and psap genes showed similar expression patterns during early development, with significant decline before early somitogenesis and stable expression thereafter. Cod pbx1a declined dramatically at late gastrula stage, while it was stable at relatively high level during larval period, while pbx1b seems to be stable throughout the early development. Cod sftpb dropped significantly before onset gastrula, thereafter, sftpb expression was stable before hatching, and abundant during larval stage. Cod sftpb was much enriched in male reproductive organ than in ovary, sftpb was also enriched in heart, spleen, head kidney, and gas gland. However, sftpb expression was weak in ovary, pancreas, brain, and liver, and gill. The phylogenetic tree showed Atlantic cod Pbx1 proteins seem to have closer evolutionary relation with coelacanth Pbx1 than ballan wrasse Pbx1, tilapia Pbx1b, or platyfish Pbx1b. Cod Psap seems to intimate with most of Actinopterygii Psap except for spotted gar Psap, and elephantfish Psap. Fish Sftpb and Psap proteins were closely clustered in the phylogenetic trees. Whale shark Sftpb and Psap may exhibit the differentiation process between these two genes in the long- term evolution history. Atlantic cod Sftpb seems to be intimate with the Sftpb existed in stickleback group XIII, tetraodon chromosome 12, Amazon molly scaffold KI519905.1, platyfish scaffold JH556662.1, and medaka chromosome 9, and their genomic environments were highly conserved as well. Highly conserved orthologous pairwise between Atlantic cod scaffold 2788 and stickleback group XIII was observed. Extensive pairwise between the sftpb paralogons in stickleback group XIII and XIV may indicate a large-scale duplication event in the evolutionary history. Key words: Atlantic cod, swim bladder, phylogeny, synteny III CONTENT Acknowledgement ........................................................................................................ II Abstract ........................................................................................................................ III Content ......................................................................................................................... IV List of Abbreviations ................................................................................................... VI List of figures ............................................................................................................ VIII List of tables ................................................................................................................. XI 1. INTRODUCTION .................................................................................................. 1 1.1 Genetic roles of surfactant- related genes ........................................................ 1 1.1.1 Functions of prosaposin and surfactant proteins ................................... 1 1.1.2 Distribution of prosaposin and surfactant B protein ............................. 3 1.1.3 Structures of prosaposin and surfactant proteins .................................. 4 1.1.4 Homologous evidences of surfactant proteins in tetrapods and teleosts. ........................................................................................................................ 6 1.2 Genetic roles of Elovl1 .................................................................................... 7 1.2.1 Structures of Elovl1 in fish ................................................................... 9 1.2.2 Transcript expression of elovl1 in Atlantic cod .................................... 9 1.3 Genetic roles of Pbx1 ....................................................................................... 9 1.3.1 Structure of zebrafish pbx1 ................................................................. 11 1.4 Early development of Atlantic cod ................................................................ 11 1.4.1 Stages of the embryonic development ................................................ 12 1.4.2 Stages of the larval development ........................................................ 12 1.4.3 Temperature of the embryonic and larval development ..................... 13 1.4.4 The importance of swim bladder in Atlantic cod................................ 13 2. MATERIAL AND METHODS ............................................................................... 15 2.1 Materials ........................................................................................................ 15 2.11 Chemicals ............................................................................................. 15 2.1.2 Equipment ........................................................................................... 15 2.2 Methods.......................................................................................................... 15 2.2.1 Preparation of experimental samples .................................................. 15 2.2.2 RNA isolation and measurement ........................................................ 16 2.2.3 cDNA synthesis and quantitative real time PCR (qPCR) ................... 17 2.2.4 Relative expression of target genes..................................................... 18 IV 2.2.5 Studies on genomic structure, phylogeny, and synteny ...................... 19 2.2.6 Data analysis ....................................................................................... 19 3. RESULTS ................................................................................................................ 21 3.1 Relative expression of target genes................................................................ 21 3.1.1 Expression of sftpb in different tissues ............................................... 21 3.1.2 Expression of key genes in swim bladder at early stages of Atlantic cod ................................................................................................................ 22 3.2 Genetic characterization, orthology, and phylogenetic analysis .................... 24 3.2.1 Atlantic cod psap ................................................................................ 24 3.2.2 Atlantic cod sftpb ................................................................................ 27 3.2.3 Atlantic cod elovl1a and elovl1b......................................................... 31 3.2.4 Atlantic cod pbx1a and pbx1b............................................................. 33 3.3 Syntenic analysis of Sftpb .............................................................................. 36 4. DISCUSSION .......................................................................................................... 39 4.1 Genetic analysis on Atlantic cod sftpb and psap ........................................... 39 4.2 Genetic analysis on Atlantic cod elovl1 ......................................................... 41 4.3 Genetic analysis on Atlantic cod pbx1 ........................................................... 41 5. conclution ................................................................................................................. 43 REFERENCE ............................................................................................................... 44 Appendix ...................................................................................................................... 54 Appendix 1: Accession NO. or transcript ID of different proteins ...................... 54 Appendix 2: Standard curve of Atlantic cod elovl1a, elovl1b, pbx1a, pbx1b, psap, and sftpb ..................................................................................................... 56 V LIST OF ABBREVIATIONS aa: Amino acids BLAST: Basic local alignment search tool BR: Brain C9: Cleavage at 9 ºC Cers: Ceramide CoAs: Coenzyme As Chro/Chr: