Title Page
Contents
Abstract 9
Introduction 11
Materials and Methods 15
Multiple-alignment of amino acid sequences of GHs 15
Homology modeling of mature form GHs 16
Synthesis, codon-optimization and cloning of fGH gene 17
Preparation of fragment for electroporation 20
Microalga strain and culture condition 21
Electroporation of C. vulgaris PKVL7422 23
Genomic DNA isolation and inserted fragment PCR 26
Western blotting assay 28
Cloning of fGH gene into E. coli BL21 (DE3) 29
Induction of pelB-linker-fGH expression 32
Purification of pelB-linker-fGH from inclusion bodies 33
Quantification of fGH expression level 35
Prediction of post-translational modifications 36
Preparation of fish feed 37
Growth-promoting effect evaluation 39
Statistical analysis 40
Results 41
Comparison of amino acid sequences 41
Comparison of putative tertiary structure of GHs 43
Selection of transformed cells using ClO2- toxicity selection marker[이미지참조] 45
Confirmation PCR of inserted fragment 47
Over-expression of pelB-linker-fGH 49
Detection and quantification of fGH expression level in C. vulgaris PKVL7422-TF1 51
Prediction of post-translational modifications in mature form of fGH 54
Growth comparison between control group and experimental group 56
Discussion 62
국문 초록 67
Reference 70
Table 1. Composition of BG11. 22
Table 2. Composition of BGPK. 25
Table 3. Primers for inserted fragment PCR 27
Table 4. Composition of fish feed. 38
Table 5. Predicted post-translational modifications in mature form of fGH. 55
Table 6. The measure indices of silver butterfly koi and discus fish. 61
Figure 1. Diagram of vectors used in this study[원문불량;p.8] 18
Figure 2. The sequence of fGH gene in CVnr_CaMV 35S::fGH_tRBCS2 vector. 19
Figure 3. Schematic diagram of the NR gene homologous recombination for transformation of C. vulgaris PKVL7422. 24
Figure 4. Diagram of pET-25b(+)-fGH vector 31
Figure 5. Conserved domains of flounder, ornamental fish and consensus GHs. 42
Figure 6. Putative tertiary structure of ornamental fish GHs and consensus GHs. 44
Figure 7. Selection results of transformed C. vulgaris PKVL7422. 46
Figure 8. PCR results of inserted fragment. 48
Figure 9. Over-expression of pelB-linker-fGH in E. coli BL21 (DE3). 50
Figure 10. Detection of fGH in C. vulgaris PKVL7422-TF1. 52
Figure 11. Western blotting of pelB-linker-fGH for fGH quantification in C. vulgaris PKVL7422-TF1. 53
Figure 12. Graph of weight and standard-length growth rate of silver butterfly koi. 57
Figure 13. Graph of weight and standard-length growth rate of discus fish. 58
Figure 14. Comparison of standard-length growth of silver butterfly koi and discus fish. 59
Figure 15. Comparison graph of weight and standard-length growth of sliver butterfly koi and discus fish. 60