표제지
국문 초록
Abstract
목차
I. 서론 18
II. 재료 및 방법 20
1. 박테리오파지 분리 20
1.1. 숙주 세균 IMCC15298 배양 20
1.2. 박테리오파지 분리실험을 위한 해수시료 채취 24
1.3. 박테리오파지의 분리를 위한 농화 (enrichment) 배양 27
1.4. 단일 파지 분리를 위한 순수분리 28
2. 박테리오파지 유전체 분석 30
2.1. 박테리오파지DNA 추출 및 제한효소 절편길이 다형성분석 (RFLP) 30
2.2. 박테리오파지 형태학적 특성 34
2.3. 박테리오파지 유전체 염기서열 결정 및 유전체 조립 35
2.4. 박테리오파지 유전체 분석 36
III. 실험 결과 37
1. 박테리오파지의 분리 37
1.1. 파지 농화 (enrichment) 배양 및 순수분리 37
1.2. 형광현미경 관찰 37
2. 제한효소 절편 길이 다형성분석 (RFLP)의 분석 결과 41
3. 박테리오파지의 형태학적 특성 43
4. 세 가지 박테리오파지의 유전체 특징 45
5. 세 가지 박테리오파지의 비교유전체 분석 51
6. 세 개의 박테리오파지의 해양 환경에서의 분포 분석 62
IV. 결론 74
V. 참고문헌 76
Table 1. The composition of LNHM media used to cultivate host bacterium IMCC15298. 22
Table 2. Physicochemical parameters of the water samples collected from the Incheon pier. 26
Table 3. Sequence information about restriction site of the HaeIII endonuclease enzyme. 33
Table 4. Genomic characteristics of P15298-A, P15298-B and P15298-C. 47
Table 5. List of reference phages genomes related to P15298-A phages, identified using GRAViTy. 53
Table 6. List of reference phages genomes related to P15298-B phages, identified using GRAViTy. 54
Table 7. List of reference phages genomes related to P15298-C phages, identified using GRAViTy. 55
Table 8. List of viral metagenomic contigs related to P15298-A, identified from the IMG/VR database. 63
Table 9. List of viral metagenomic contigs related to P15298-C, identified from the IMG/VR database. 64
Figure 1. Map of the sampling site of this study (Incheon Yeonan pier in South Korea).-37° 26'58.7"N 126° 35'34.6"E 25
Figure 2. A research scheme of this study. It is a P15298 bacteriophage study conducted in this study. 29
Figure 3. Growth curve of host bacteria co-cultured with the isolated bacteriophage. This is the growth curve measured by a flow cytometer after inoculation of enriched... 38
Figure 4. Growth curve of host bacteria co-cultured with the isolated bacteriophages in a large volume cultivation. Samples were measured by a flow... 39
Figure 5. Epifluorescence microscope image of bacteriophage isolated in this study. (a) Host strain IMCC15298 and infected bacteriophage P15298. Red arrow indicates... 40
Figure 6. Restriction fragment length polymorphism analysis of the extracted phage DNA. In the gel electrophoresis picture, the first lane indicates a 100 bp of DNA... 42
Figure 7. Transmission electron micrographs of bacteriophages isolated in this study. The morphology was confirmed by a transmission electron microscopy. The... 44
Figure 8. Assembly graphs of three phage genome sequences. Genomic sequences were submitted to Bandage program to predict assembled genomic contigs. 48
Figure 9. Genomic map of P15298-A, P15298-B, and P15298-C. (a) P15298-A, (b) P15298-B (c) P15298-C. Genomic map was created by Easyfig program referred to... 49
Figure 10. Sequencing coverages of the three phage genomes. The y-axis is sequencing coverage, and the x-axis is length (bp). (a). P15298-A, (b). P15298-B, (c). P15298-C. 50
Figure 11. Viral proteomic tree of the three bacteriophages. Based on the results of analysis by VipTree programs. (a). P15298-A, (b). P15298-B, (c). P15298-C. 52
Figure 12. Dendrogram showing the clustering of P15298-A with other isolated phage genomes. Phylogenetic analysis was conducted by GRAViTy programs... 56
Figure 13. Dendrogram showing the clustering of P15298-B with other isolated phage genomes. Phylogenetic analysis was conducted by GRAViTy programs... 57
Figure 14. Dendrogram showing the clustering of P15298-C with other isolated phage genomes. Phylogenetic analysis was conducted by GRAViTy programs... 58
Figure 15. Genomic similarities between bacteriophage P15298-A and other isolated phage genomes. Comparison of intergenomic similarity based on... 59
Figure 16. Genomic similarities between bacteriophage P15298-B and other isolated phage genomes. Comparison of intergenomic similarity based on... 60
Figure 17. Genomic similarities between bacteriophage P15298-C and other isolated phage genomes. Comparison of intergenomic similarity based on... 61
Figure 18. Blast-based genome sequence comparison between P15298-A and viral metagenomic contigs from the Delaware Bay and the North Sea. (a) Delaware Bay,... 65
Figure 19. Blast-based genome sequence comparison between P15298-A and viral metagenomic contigs from the Fram Strait and the Columbia River. (a) Fram... 66
Figure 20. Blast-based genome sequence comparison between P15298-A and viral metagenomic contigs from the Southern Ocean and the California coast. (a)... 67
Figure 21. Blast-based genome sequence comparison between P15298-A and viral metagenomic contigs from the Saanich Inlet, Botany Bay, and the Atlantic... 68
Figure 22. Blast-based genome sequence comparison between P15298-C and viral metagenomic contigs from the Pacific Ocean and the Georgia coast. (a)... 69
Figure 23. Blast-based genome sequence comparison between P15298-C and viral metagenomic contigs from the North Sea and the Chesapeake Bay. (a)... 70
Figure 24. Blast-based genome sequence comparison between P15298-C and viral metagenomic contigs from the Port Hacking. 71
Figure 25. Sequence similarities between P15298-A and marine viral metagenomic contigs. The highest similarity is Delaware Bay 51.6%, North Sea... 72
Figure 26. Sequence similarities between P15298-C and marine viral metagenomic contigs. The highest similarity was Pacific at 44.4% and... 73