초록 ▼

Ⅳ. 연구개발결과
○ 동충하초 유전체의 생물정보학적 분석 시스템 구축
- 동충하초 유전체 데이터의 de novo assembly, gene prediction, annotation을 위한 표준 분석 파이프라인 확립
- 이차대사산물 생합성 유전자 집단 추출을 위한 분석 방법론 확보
- 전사체 데이터 분석을 위한 파이프라인 구축
○ 육좌균목 균류에서 독성 이차대사산물 (beauvericin, bassianolide) 생합성 유전자의 진화 및 분포 추정
- 도메인 분석과 adenylation 염기서열 기반

Ⅳ. 연구개발결과
○ 동충하초 유전체의 생물정보학적 분석 시스템 구축
- 동충하초 유전체 데이터의 de novo assembly, gene prediction, annotation을 위한 표준 분석 파이프라인 확립
- 이차대사산물 생합성 유전자 집단 추출을 위한 분석 방법론 확보
- 전사체 데이터 분석을 위한 파이프라인 구축
○ 육좌균목 균류에서 독성 이차대사산물 (beauvericin, bassianolide) 생합성 유전자의 진화 및 분포 추정
- 도메인 분석과 adenylation 염기서열 기반 계통분석을 통해 gene fusion이 동충하초 속분화 이전 시점에 발생하였음을 규명
- 육좌균목 균류 내 유일하게 노랑다발동충하초만이 두 가지 독성 생산 가능함 규명
- beauvericin 생합성 유전자는 동충하초과 균류의 공통조상으로부터 유래한 반면 bassianolide 생합성 유전자는 근래에 horizontal gene transfer에 의해서노랑다발동충하초의 유전체로 도입되었을 가능성 제시
○ 색소형성을 조기에 할 수 있는 붉은자루동충하초 균사체 조제 및 배양방법 개발
○ 대사체 기술을 활용한 동충하초 종별(노랑다발동충하초, 붉은자루동충하초, 번데기동충하초) 대사과정 및 대사 생성물에 대한 연구
- 동충하초 종별 대사체 차이성 규명 및 동충하초의 생물소재 발굴

Abstract ▼

Cordyceps is an entomopathogenic fungus belonging to the family Cordycepitaceae. As an immunity-boosting food, Cordyceps has been widely used the East, together with ginseng and deer antlers. A number of pharmacological researches have reported that Cordyceps spp. have anti-inflammatory, anti-cancer

Cordyceps is an entomopathogenic fungus belonging to the family Cordycepitaceae. As an immunity-boosting food, Cordyceps has been widely used the East, together with ginseng and deer antlers. A number of pharmacological researches have reported that Cordyceps spp. have anti-inflammatory, anti-cancer, anti-oxidant, antiviral effects. Analyzing genome data is the basis of finding useful secondary metabolite synthetic genes and understanding the related metabolic pathways. Researches on fungal secondary metabolite synthetic genes have been scarcely performed in Korea. Establishing systematic and comprehensive strategy for omics research is necessary to find new biomaterials adding higher value on Cordyceps. In this study, we conducted genomic data mining of Cordyceps spp., especially, C. militaris, C. bassiana, and C. pruinosa.
The development stages of C. bassiana fruiting bodies were divided as like before perithecium formation(1), beginning formation(2), forming(3) and late formation(4). The highest total phenol compounds were found at stage 3. Transcriptome analysis revealed that gene expression differed among the stages. 8553 genes were estimated from C. bassiana and they were sorted into 5 clusters based on their level of expression. C. bassiana genes were regulated differently in light and dark condition. The gene related to toxin, antibiotics and metal ion activation genes were induced in dark condition. Secondary metabolic genes were up-regulated in light conditions.
C. pruinosa grew fast on pH 8 on SDAY and slow on pH 4 on SDAY. Optimal growth was at 25℃. pH and temperature were not involved in pigmentation. C. pruinosa produced red pigment only in light condition. Transcriptome analysis of mycelium grown on SDAY in light/dark condition for 3 days showed that secondary metabolites compounds synthesis related genes were up-regulated in light condition.
C. pruinosa spores were formed 1000 fold in light condition than in dark condition. When C. pruinosa grew on SDAY in each light and dark condition for 3, 5 and 14 days, the red pigment were produced only in light condition. Spore formation was more enhanced in light condition than dark condition. Transcriptime comparison between 5 days versus 14 days cultivation showed that genes involved in transportation were up regulated and gliotoxin gene was down regulated. Allergen Asp F4 gene was up regulated in dark condition. C. pruinosa has 9 PKS genes and 6 NRPS genes. 4 PKS genes and 1 NRPS gene were regulated by light.
Gene knock out system for C. pruinosa Cordyceps was established using Agrobacterium-mediated transformation technique. A NRPS gene of C. pruinosa regulated by light was studied in detail through gene mutation. The mutant produced more dense red pigment in light condition. Thus, the gene is assumes to be a gene involved in the control of pigment synthesis. Overall, the analysis of transcriptomes contributed to make a research infrastructure for the utilization of Cordyceps as valuable resources.
The main purpose of this study is to develop functional materials and to investigate the correlation between metabolomics and other field by analysing metabolites. To comprehend biological system, it is important to understand not only each component of genome, transcriptome, proteome and metabolome but also their interactions. Especially, metabolomics, which has a direct impact on phenotype and function of organisms, is a useful tool to investigate feature of organisms represented by biochemical phenomenon. Metabolome analysis of Cordyceps with NMR, GC-MS, HPLC instrument coupled with multivariate statistical data was performed. Metabolome analysis of Cordyceps based on culture condition (Light/ Dark/ Lipid/ SDAY-lipid, Fe, N) and their developmental stage was also carried out. Antioxidant activity assay was determined based on the DPPH radical scavenging activity and total phenolic content (TPC). Anti-cancer activity assays were conducted by MTT and NO assays.
Cordyceps bassiana strains cultivated in dark and light condition represented higher levels of sugar, glutamic acid, serine, proline and ornithine than those cultivated in the other conditions. Erythritol and linoleic acid were detected in Fe condition and Lipid condition, respectively. In perithecium formation stage, the levels of amino acids, malic acid and fatty acid were higher than other stages. Total phenolic content was also highest in perithecium formation stage. Therefore, perithecium formation stage has a good antioxidant activity rather than other stages.
PCA score plot of Cordyceps pruinosa showed a clear separation between samples cultivated in N condition and other conditions. Antioxidant activity was also highest in N condition. Beauvericine, a functional materials, was increased in SD, SL and FD conditions. Cordyceps militaris strains obtained in N, Lipid, Light and Fe condition were clearly separated in PCA score plot. According to development stage, the levels of amino acids were increased, while those of organic acids were decreased. Codycepin, a physiological active substance, was increased according to their developmental stages.
Although culture condition was identical, metabolite profile was observed differently according to strain. Therefore, it is important to select proper strain for investigation of functional materials.