애기뿔소똥구리의 진균 억제 공생미생물의 규명
Characterization of symbiotic bacteria associated with hygiene of the dung beetle Copris tripartitus
- 주제(키워드) Dung beetle , Copris tripartitus , Actinobacteria , PCR-DGGE , Antifungal activity
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 손영창, 권학철
- 발행년도 2015
- 학위수여년월 2015. 2
- 학위명 박사
- 학과 및 전공 일반대학원 KIST강릉분원학.연협동과정
- 세부전공 분자생물학, 천연물화학
- 원문페이지 vii, 96
- 실제URI http://www.dcollection.net/handler/kangnung/000000007059
- 본문언어 영어
초록/요약
Dung beetle, Copris tripartitus is a native species in Korea. They are saprophagous beetles using dung for feeding and breeding in soil environment. Therefore, the dung beetle can be easily exposed to the threat of invasion by pathogenic microbes. However, the dung beetles defend their offspring by providing brood care to brood ball including egg or larva. These suggest that the dung beetles may have defensive strategies such as symbiosis with bacteria producing antimicrobial substances. In this study, we specified symbiotic bacteria associated with hygiene of dung beetle by culture-dependent, culture-independent and chemical approaches. Total of 120 bacterial isolates from dung beetle and their brood ball were screened for antifungal activity by dual culture technique against ubiquitous soil fungi (Aspergillus fumigatus and Aspergillus flavus) and entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae). Sixteen bacterial isolates showed antifungal activity. Among these, the strain AT57 exhibited strong antifungal activity against all tested fungi and produced sceliphrolactam as secondary metabolites. In culture-independent approach by PCR-DGGE, bacterial communities associated with dung beetle were profiled and characterized. The DGGE bands corresponding to the strain AT57 was detected in all dung beetles and brood balls.
more목차
Introduction 1
Objectives 6
Chapter 1 7
1.1 Introduction 8
1.2 Materials & Methods 12
1.2.1 Sample collection 12
1.2.2 Microbial isolation 13
1.2.3 Primary antifungal screening by the dual culture technique 14
1.2.4 Molecular identification and phylogenetic analysis of bacterial strains 16
1.2.5 Molecular identification of fungal strains 17
1.2.6 Antifungal assay against fungal strains isolated from dung beetle 18
1.3 Results and Discussion 19
1.3.1 Microbial isolation 19
1.3.2 Primary antifungal screening by the dual culture technique 20
1.3.3 Antifungal activity against fungi isolated from dung beetles and brood balls 23
1.3.4 Molecular identification and phylogenetic analysis of antifungal bacterial strains 26
Chapter 2 31
2.1 Introduction 32
2.2 Materials & Methods 36
2.2.1 Microbial DNA extraction 36
2.2.2 Nested PCR amplification for DGGE analysis 37
2.2.3 DGGE marker production 45
2.2.4 Denaturing Gradient Gel Electrophoresis (DGGE) 45
2.2.5 Band excision & Identification 46
2.2.6 Chemical analysis of secondary metabolites of the strain AT57 46
2.3 Results and Discussion 47
2.3.1 Microbial DNA extraction and purification from dung beetle and brood ball 47
2.3.2 Nested PCR amplification for DGGE analysis 49
2.3.2.1 Bacteria group 49
2.3.2.2 Actinobacteria group 50
2.3.2.3 Antifungal strain group 53
2.3.3 DGGE marker production 56
2.3.4 Bacterial communities of the dung beetle by DGGE 57
2.3.4.1 Bacteria group 57
2.3.4.2 Actinobacteria group 61
2.3.4.3 Antifungal strain group 64
2.3.5 Antifungal compound produced by strain AT57 68
Conclusions 72
Suggestion for further works 74
References 75
Appendixes 92
