Title Page
Contents
요약 10
CHAPTER 1. General Introduction 11
1.1. Target species 12
1.2. Appetite 13
1.3. Ghrelin 14
1.4. Key research needs and objective of this study 15
CHAPTER 2. Molecular characterization of ghrelin in starry flounder (Platichthys stellatus) 17
2.1. Introduction 18
2.2. Gene structure 20
2.2.1. Materials and methods 20
2.2.2. Results and discussions 25
2.3. Multiple alignment and phylogenetic tree 29
2.3.1. Materials and methods 29
2.3.2. Results and discussions 30
2.4. Tissue distribution 36
2.4.1. Materials and methods 36
2.4.2. Results and discussions 39
2.5. Conclusion 41
CHAPTER 3. Expression characteristics of ghrelin in starry flounder (Platichthys stellatus) 42
3.1. Introduction 43
3.2. Starvation and ghrelin trigger 46
3.2.1. Materials and methods 46
3.2.2. Results and discussions 49
3.3. Effect of salinity 58
3.3.1. Materials and methods 58
3.3.2. Results and discussions 61
3.4. Effect of ghrelin recombinant on feed intake 69
3.4.1. Materials and methods 69
3.4.2. Results and discussions 73
3.5. Conclusion 84
References 85
Table 1. Primer used for sequencing and RACE PCR of ghrelin gene 24
Table 2. Ghrelin sequences in other vertebrates used for multiple alignment and mature peptide's nucleotide sequences similarity with starry flounder 32
Table 3. Primers used for RT-qPCR of ghrelin gene 38
Figure 1. Nucleotide and deduced amino acid sequences of the complete ghrelin cDNA in starry flounder. The numbers on the left and right sides... 26
Figure 2. Schematic diagram illustrating the exon/intron organization of the starry flounder ghrelin gene. The diagram shows the predicted process by... 28
Figure 3. Multiple alignment of the deduced amino acid sequence of starry flounder ghrelin with those of other vertebrates using ClustalW. The... 33
Figure 4. Phylogenetic tree based on the nucleotide sequence of the mature peptide of starry flounder ghrelin and those of other vertebrate species.... 35
Figure 5. Relative ghrelin expression, normalized to GAPDH, in various tissues of starry flounder. The vertical bars represent the mean ± SE (n=... 40
Figure 6. The dry weight of stomach contents at different time points after feeding. The vertical bars represent the mean ± SE (n=12). Different... 50
Figure 7. Images of gastrointestinal tract at different time points after feeding. The gastrointestinal tract is depicted horizontally, starting from the... 51
Figure 8. Relative ghrelin expression normalized to GAPDH after 24 hours of starvation. The vertical bars represent the mean ± SE (n=6). Ghrelin... 52
Figure 9. Relative ghrelin expression normalized to GAPDH in response to starvation for 96 hours. The vertical bars represent the mean ± SE (n=... 55
Figure 10. Gastrointestinal tract images after 96 hours of starvation. The gastrointestinal tract is displayed horizontally from left to right, showing... 56
Figure 11. Histological organization of the liver of starry flounder during 96 hours of starvation. A. Immediately after feeding. B. 48 hours after... 57
Figure 12. Histological organization of the intestine of starry flounder during 96 hours of starvation. A. 48 hours after feeding. B. 96 hours after... 57
Figure 13. Salinity and refeeding experiment tank setting illustration. 60
Figure 14. Dry weight of stomach contents in the control, 15 psu, and 5 psu groups at different time intervals after feeding. The vertical bars... 62
Figure 15. Gastrointestinal tract images of all salinity groups at different time intervals after feeding. The gastrointestinal tract in the picture is... 63
Figure 16. Relative ghrelin expression normalized to GAPDH in starry flounder in the 32 psu group at different time intervals after feeding. The... 66
Figure 17. Relative ghrelin expression normalized to GAPDH in starry flounder in the 15 psu group at different time intervals after feeding. The... 67
Figure 18. Relative ghrelin expression normalized to GAPDH in starry flounder in the 5 psu group at different time intervals after feeding. The... 68
Figure 19. Ghrelin recombinant injection experiment tank setting illustration. 72
Figure 20. Growth hormone concentration in brain tissue of saline-injected starry flounder. The vertical bars represent the mean ± SE (n=6). The... 74
Figure 21. Growth hormone concentration in brain tissue of ghrelin-injected starry flounder. The vertical bars represent the mean ± SE (n=6). The... 75
Figure 22. Initial body weight (g) of control and ghrelin-injected starry flounder. The vertical bars represent the mean ± SE (n=20). The saline... 77
Figure 23. Daily feed intake of control and ghrelin-injected starry flounder. The vertical bars represent the mean ± SE (n=20). The saline (0.8%)... 78
Figure 24. Feed efficiency (%) of control and ghrelin-injected starry flounder. The vertical bars represent the mean ± SE (n=20). The saline... 80
Figure 25. Final body weight (g) of control and ghrelin-injected starry flounder. The vertical bars represent the mean ± SE (n=20). The saline... 81
Figure 26. Weight gain (%) in control and ghrelin-injected starry flounder. The vertical bars represent the mean ± SE (n=20). The saline (0.8%)... 82
Figure 27. Specific growth rate (%) in control and ghrelin-injected starry flounder. The vertical bars represent the mean ± SE (n=20). The saline... 83