CHARACTERISATION, DIVERSITY AND EXPRESSION PATTERNS OF MUCIN AND MUCIN-LIKE GENES IN SEA ANEMONES Alaa Abdulgader Haridi Bachelor of Biology, Master of Plant Protection Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Earth, Environment and Biological Sciences Science and Engineering Faculty Queensland University of Technology 2018 Keywords Actinia tenebrosa, Actinaria, Air exposure, Anthozoan, Aulactinia veratra, Cnidarians, De novo assembly, Gel-forming mucins, Glycoproteins, Mucus, Mucin, Mucin-like, Mucin gene diversity, Mucin genes expression, Mucin5B-like, Mucin1- like, Mucin4-like, quantitative real-time PCR, RNA-sequences, Sea anemone, Transcriptome, Transmembrane-mucins, Trefoil peptide Characterisation, diversity and expression patterns of mucin and mucin-like genes in sea anemones i Abstract The waratah sea anemone, Actinia tenebrosa, inhabits the intertidal zone of Australia and New Zealand. This environment exposes A. tenebrosa to a myriad of abiotic and biotic challenges including heat, desiccation and pathogenic microorganisms. Actinia tenebrosa is thought to respond to some abiotic and biotic challenges by producing a thick covering of protective mucus. The proteinaceous scaffold that supports this protective mucus covering, are mucin glycoproteins, which are encoded by a range of mucin genes. Earlier research into cnidarians generated explanations of the significance of mucus in cnidarian immunity, but little research has been undertaken to investigate the mucin gene repertoire of cnidarians and no studies have evaluated mucin gene expression patterns under environmental challenges. As a consequence, the first objective of this research project was to use RNA-sequencing, De novo assembly and annotation to identify the mucin and mucin-like genes present in a range of cnidarian species. Using this approach, we were able to identify a range of gel-forming and transmembrane mucin genes in A. tenebrosa, including full-length mucins (mucin1-like and mucin4-like), as well as partial-length mucins (mucin5B- like, mucin6-like and mucin3A-like), a range of mucin-like genes, and mucin associated genes. The domain structure of the identified full-length mucin genes was found to be similar to that of the homologous genes in other species, and the majority of the mucin genes were found to be present in the other cnidarian species examined. The second objective of this research was to investigate the expression of the mucin genes under an environmental challenge. Specifically, we examined genome wide patterns of gene expression in two intertidal sea anemones, Aulactinia veratra and A. tenebrosa under an aerial exposure treatment to test the hypothesis that mucin Characterisation, diversity and expression patterns of mucin and mucin-like genes in sea anemones ii genes will have significantly higher expression under this treatment. Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra, but none of the identified mucin genes were differentially expressed in A. tenebrosa. These findings indicate that mucin genes are not expressed in the same way in these two intertidal anemone species under aerial exposure treatments. The results from this research demonstrate that there is a diverse repertoire of mucin genes in cnidarians and that the majority of these genes are widespread across this phylum. The expression patterns of these mucin genes were varied across the two test species and, in many cases, did not conform to our hypothesis, with the exception of mucin4-like and mucin5B-like in A. veratra. These findings establish a baseline from which future research can extend our understanding of mucin genes and proteins in phylum Cnidaria. The discovery of a diverse range of mucin genes in sea anemone species provided a basic reference for future mucin studies in cnidarians, but also could lead to research into their application in the pharmacological, clinical and cosmetic industries. Characterisation, diversity and expression patterns of mucin and mucin-like genes in sea anemones iii Table of Contents Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii Table of Contents .................................................................................................................... iv List of Figures ........................................................................................................................ vii List of Tables ............................................................................................................................ x List of Abbreviations ............................................................................................................... xi Statement of Original Authorship .......................................................................................... xii Acknowledgements ............................................................................................................... xiii Chapter 1: Introduction ...................................................................................... 1 1.1 Background, knowledge gap and research problem ....................................................... 1 1.2 Research aim .................................................................................................................. 2 1.3 Research objectives ........................................................................................................ 2 1.4 Research methods .......................................................................................................... 3 1.5 Research significance ..................................................................................................... 3 Chapter 2: Literature review .............................................................................. 5 2.1 Overview ........................................................................................................................ 5 2.2 Mucus ............................................................................................................................. 5 2.3 Mucin ............................................................................................................................. 6 2.4 Actinia tenebrosa and Aulactinia veratra .................................................................... 12 2.5 Conclusion ................................................................................................................... 15 Chapter 3: General methods ............................................................................. 19 3.1 Overview ...................................................................................................................... 19 3.2 Sample collection and maintenance ............................................................................. 19 3.3 RNA isolation .............................................................................................................. 20 3.4 Library preparation, RNA-Sequencing and quality control ......................................... 20 3.5 Transcriptome assembly using the De Novo assembler and assessment...................... 21 3.6 Transcriptome functional annotation and gene ontology ............................................. 21 3.7 Mucin and mucin-like candidate identification ............................................................ 21 3.8 Read mapping and differential gene expression analysis ............................................. 22 3.9 Gene set enrichment analysis (GSEA) ......................................................................... 22 Chapter 4: Identification, diversity and domain structure analysis of mucin and mucin-like genes in sea anemone Actinia tenebrosa ....................................... 23 4.1 Introduction .................................................................................................................. 24 4.2 Materials and methods ................................................................................................. 25 Characterisation, diversity and expression patterns of mucin and mucin-like genes in sea anemones iv 4.3 Results .......................................................................................................................... 27 4.4 Discussion ..................................................................................................................... 39 4.5 Conclusion .................................................................................................................... 42 Chapter 5: The expression of mucin-like genes using qRT-PCR .................. 43 5.1 Introduction .................................................................................................................. 43 5.2 Materials and methods .................................................................................................. 44 5.3 Results .......................................................................................................................... 46 5.4 Discussion ..................................................................................................................... 47 Chapter 6: The influence of aerial exposure on Aulactinia veratra mucin gene expression using the