声明
List of abbreviations
Chapter 1 Literature review
1 Overview of nuclear receptor superfamily
1.1 Members of nuclear receptor superfamily and their unified nomenclature
1.2 Structure of nuclear receptor
1.3 Function of nuclear receptor
1.4 Research progress of Nr2f2
2 Gametogenesis in vertebrates
2.1 Testis and spermatogenesis
2.2 Ovary and oogenesis
3 Purpose and significance of this study
Chapter 2 Isolation and expression of nr2f2 in Nile tilapia
1 Introduction
2 Materials and methods
2.1 Experimental animal Scientific classification
2.2 Main instruments and reagents
2.3 Gene cloning
2.4 Construction of phylogenetic tree
2.5 Syntenic analysis
2.6 RT-PCR and Real-time PCR
2.7 Transcriptome analyses
2.8 In situ hybridization
2.9 Antibody preparation
2.10 Western blot
2.11 Immunofluorescence
3 Results
3.1 Cloning of nr2f2a and nr2f2b in Nile tilapia
3.2 Amino acid sequence alignment of Nr2f2a and Nr2f2b
3.3 Identification of nr2f2s in vertebrates
3.4 Phylogenetic analysis of nr2f2 in vertebrates
3.5 Syntenic analysis of nr2f2 in vertebrates
3.6 Tissue distribution of nr2f2a and nr2f2b in Nile tilapia
3.7 Ontogenic expression of nr2f2a and nr2f2b in Nile tilapia gonad
3.8 Cellular localization of nr2f2a and nr2f2b by ISH
3.9 Verification of Nr2f2a and Nr2f2b specific antibodies by Western blot
3.10 Cellular localization of Nr2f2a by Immunofluorescence
4 Discussion
4.1 nr2f2a and nr2f2b originated from the fish specific whole genome duplication
4.2 nr2f2b experienced a secondary loss in some basal group teleosts
4.3 The expression of nr2f2a and nr2f2b in Nile tilapia
Chapter 3 Functional study of nr2f2s in Nile tilapia
1 Introduction
2 Materials and methods
2.1 Experimental animal
2.2 Main instruments and reagents
2.3 Inhibitor administration
2.4 Target site designing for mutation
2.5 Synthesis of gRNA and Cas9 mRNA and microinjection
2.6 Mutation detection and establishment of mutant line
2.7 Egg and sperm quality assessment
2.8 Embryonic development observation
3 Results
3.1 Inhibition of nr2f2 resulted in aberrant gametogenesis in Nile tilapia
3.2 Disruption of nr2f2a and nr2f2b by CRISPR/Cas9
3.3 Establishment of nr2f2a and nr2f2b mutant lines
3.4 Haploinsufficiency of nr2f2 in female Nile tilapia resulted in decreased egg quality
3.5 Offspring of nr2f2 female heterozygotes displayed aberrant embryogenesis
4 Discussion
4.1 Both nr2f2a and nr2f2b are involved in Nile tilapia oogenesis
4.2 nr2f2a is critical while nr2f2b is dispensable for Nile tilapia oogenesis
4.3 nr2f2a is important for Nile tilapia testis development and spermatogenesis
Chapter 4 Conclusions
参考文献
致谢
Publications during Ph.D. study
Research activities during Ph.D. study
西南大学;
