초록 ▼

이 과제에서는 국내 수집 균주들의 주요 곰팡이 독소 생성 기능 변이조사 및 특성 분석을 하고 변이집단 간 독소 생합성 대사 관련 효소 유전자 및 조절 관련 유전자의 발현을 비교, 분석하고자 한다. 한편 후자린산 생합성유전자 발굴을 위해 고생산 국내 F . oxysporum를 확보하고 REMI 돌연변이 유도법 최적화를 통해 형질전환체를 육성하며 형질전환체 스크리닝을 통해 후자린산 미생성 균주를 선발하면 해당유전자의 분자유전학적 특성을 규명한다. 유전집단의 연구를 위해 다년간 곡류시료를 수집하고 Fusarium 균주를 분리하여 동정한

이 과제에서는 국내 수집 균주들의 주요 곰팡이 독소 생성 기능 변이조사 및 특성 분석을 하고 변이집단 간 독소 생합성 대사 관련 효소 유전자 및 조절 관련 유전자의 발현을 비교, 분석하고자 한다. 한편 후자린산 생합성유전자 발굴을 위해 고생산 국내 F . oxysporum를 확보하고 REMI 돌연변이 유도법 최적화를 통해 형질전환체를 육성하며 형질전환체 스크리닝을 통해 후자린산 미생성 균주를 선발하면 해당유전자의 분자유전학적 특성을 규명한다. 유전집단의 연구를 위해 다년간 곡류시료를 수집하고 Fusarium 균주를 분리하여 동정한 후 독소생성능을 분석한다. 각 균주집단은 기존 분자 마커 및 독소의 생성관련 유전자의 염기서열 이용하여 균주 집단 내 또는 집단 간 변이분석을 통한 유전적다양성 분석을 실시한다. 균주 특이적 분자마커를 개발하고 집단 분석에 활용한다.

Abstract ▼

In order to find genetic difference between deoxynivalenol (DON) and nivalenol (NIV) producing Fusaria, Fusarium graminearum that produce DON and F. asiaticum that produce NIV were compared for expression of a few trichothecene biosynthetic genes. Two strains of each chemotype were inoculated in ric

In order to find genetic difference between deoxynivalenol (DON) and nivalenol (NIV) producing Fusaria, Fusarium graminearum that produce DON and F. asiaticum that produce NIV were compared for expression of a few trichothecene biosynthetic genes. Two strains of each chemotype were inoculated in rice medium or liquid medium and grown for 21 days. At 2-day intervals, a portion of the culture was collected for toxin analysis and RNA isolation. Toxin amount measured by GC/MS showed that toxin production was different by culture method with rapid detection in liquid culture. NIV production was generally much lower than DON's. When boosted for trichothecene production, however, NIV strain produced NIV as much as DON. Analysis of gene expression by real-time PCR was performed with 8 genes for farnesyl phyrophosphate synthase (FPP) and trichothecene biosynthesis including TRI4, TRI5, TRI6, TRI10, TRI11, TRI12, and TRI101. Comparison of gene expression between DON and NIV producers showed that toxin amount was proportional to gene expression. Northern analysis confirmed this result. TRI5, TRI10, TRI11, TRI12 and TRI101 genes were expressed earlier and stronger in NIV strain. Expression of TRI6, TRI4 and FPP genes, however, were generally more in DON strain. This result did not explain difference between DON and NIV isolates, suggesting involvement of other factor(s). To compare promoter activity between these two types, the TRI5 promoter of DON and NIV isolates were attempted to be swapped. Swapping by knock-out and replacement with a antibiotic resistant gene is in progress.
Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been found or isolated. In order to explore FA biosynthetic gene(s), knock out of the gene(s) by using restriction enzyme mediated insertional (REMI) mutagenesis has been applied to FA producing F.oxysporum strains. REMI of two stains tested has yielded more than 7,100 mutants with efficiencies at least 3.1%. To set-up mutant screening system using phytotoxicity of FA, various grains and vegetable seeds were tested for germination in cultures where FA-producing strain was grown. Among eleven seeds, Chinese cabbage seeds were selected for further screening of the REMI mutants. Each mutant isolate was grown in Czapek-Dox broth at 25C with vigorous shacking for 3 weeks then culture filtrate was filtered into a petri dish containing 20 cabbage seeds. Each container was incubated at 25C under continuous fluorescence light and monitored for seed germination at 24 and 48 hours. Among about 4,000 mutants tested, about 50 appeared that the cabbage seeds were germinated at 48 hours. As FA is revealed as a polyketide recently, we have attempted to identify a polyketide synthase (PKS) gene cluster for FA biosynthesis in FA producing F. oxysporum isolate.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 6
• 제1장 서론 ... 9
• 제2장 국내외 기술개발 현황 ... 11
• (1) 세계적 수준 ... 11
• (2) 국내수준 ... 11
• 제3장 연구개발수행 내용 및 결과 ... 12
• 제1절 국내 우점 F usarium g raminearum 집단의 독소생성능변이에 대한 분자유전학적 연구 ... 12
• 제2절 후자린산 독소 생합성유전자 발굴 ... 22
• 제3절 독소 생성 F usarium 곰팡이의 유전집단 특성 분석 ... 28
• 제4장 연구개발목표 달성도 및 대외기여도 ... 64
• 제1절 목표대비 달성도 ... 64
• 제2절 정량적 성과 ... 64
• 제5장 연구개발결과의 활용계획 ... 69
• 제6장 중요 변동사항 ... 70
• 제7장 참고문헌 ... 72
• 참고 1. 연차평가 지적사항 및 조치결과 ... 74
• 참고 2. 주요연구 성과 ... 75
• 주요 결과 요약서 ... 76