Title Page
Contents
Abstract 9
1. Introduction 11
1.1. General introduction 11
1.2. Target genes 16
1.2.1. Catalase (CAT) 16
1.2.2. Glutathione peroxidase (GPx) 16
1.2.3. Glutathione-s-transferase (GST) 17
1.2.4. Superoxide dismutase (SOD) 18
1.3. Objectives of the study 19
2. Materials and methods 20
2.1. Target genes sequences and RNA isolation. 20
2.2. Bioinformatics sequence analysis and multiple sequence alignment 21
2.3. Fish and laboratory management 21
2.4. In vivo exposure of fish to ammonia stress 22
2.5. Semi quantitative PCR analysis 23
3. Results 25
3.1. Target genes sequences and RNA isolation 25
3.2. Multiple sequence alignment and bioinformatics sequence analysis 32
3.3. Semi quantitative PCR results 42
4. Discussion 50
5. Conclusion 55
6. References 56
Table 1. Primers used in this study 31
Figure 1. Full length sequence of O. dancena CAT gene. The ORF is underlined and Poly-... 27
Figure 2. Full length sequence of O. dancena GPx gene. The ORF is underlined and grey... 28
Figure 3. Full length sequence of O. dancena GST gene. The ORF is underlined and grey... 29
Figure 4. Full length sequence of O. dancena SOD gene. The ORF is underlined and... 30
Figure 5. Multiple sequence alignment of CAT gene of marine medaka fish and other... 34
Figure 6. Multiple sequence alignment GPx gene of marine medaka fish with other close... 36
Figure 7. Multiple sequence alignment GST gene of marine medaka fish with other close... 37
Figure 8. Multiple sequence alignment SOD gene of marine medaka fish with other close... 38
Figure 9. Phylogenetic tree of marine medaka with other 5 teleost species. A shows... 40
Figure 10. Phylogenetic tree of marine medaka with other 5 teleost species. A shows... 41
Figure 11. CAT expression result of adult medaka fish gills and kidney. 43
Figure 12. GPx expression result of adult medaka fish gills and kidney. 44
Figure 13. GST expression result of adult medaka fish gills and kidney. 45
Figure 14. SOD expression result of adult medaka fish gills and kidney. 46
Figure 15. CAT expression of whole the body juvenile medaka fish exposed to different... 48
Figure 16. GPx expression of whole the body juvenile medaka fish exposed to different... 48
Figure 17. GST expression of whole the body juvenile medaka fish exposed to different... 49
Figure 18. SOD expression of whole the body juvenile medaka fish exposed to different... 49