초록

○ 연구결과
-Bioinformaticstools을 이용한 유전자 선발
-벼도열병균의 발달 단계별 RNA 추출
-qRT-PCR을 이용한 유전자 발현 조사
-결실돌연변이체를 생성하기 위한 construct제작
̵-MultiplexPCR을 이용한 결실돌연변이체 screening
-Southernblot과 RT-PCR
̵-복원형질전환체를 생성하기 위한 construct제작
-MultiplexPCR을 이용한 복원형질전환체 screening
̵-유전자의 기능을 벼도열병균의 생활사에서 분석

Abstract ▼

Magnaporthe oryzae is one of the most destructive fungal pathogen to rice. It has emerged as a model organism for the characterization of molecular mechanism relevant to pathogenic development in host plants. Similar to other pathogenic fungi, conidiation plays a central role in initiation of M. ory

Magnaporthe oryzae is one of the most destructive fungal pathogen to rice. It has emerged as a model organism for the characterization of molecular mechanism relevant to pathogenic development in host plants. Similar to other pathogenic fungi, conidiation plays a central role in initiation of M. oryzae infection and spread over a large area. However, relatively little is known regarding the molecular mechanisms that underlie conidiation in M. oryzae. As a first step to understand conidiogenesis in this fungus, we measured genome-wide gene expression profiles during conidiation using a whole genome oligonucleotide microarray. At a two-fold expression difference, approximately 4.42% and 4.08% of genes were upregulated and downregulated, respectively, during conidiation.
Transcriptional regulation is an important process for development and pathogenicity in response to environmental factors. Therefore, transcription factors (TF_s) is critical for understanding regulation of fungal development and pathogenicity. In M. oryzae, 481 transcription factors(TF_s) were identified from fungal transcription factor database, but only about 24 TF genes were functionally characterized.
AT-hook motif-containing DNA-binding proteins have not been characterized in fungi.
The AT-hook is a small DNA-binding protein motif which was first described in the high mobility group non-histone chromosomal protein HMG-I(Y). The AT-hook binds to the minor groove of adenine-thymine (AT) rich DNA. Recently, we identified 23 AT-hook motif genes (MoATh01 to MoATh23) in the genome of M. oryzae. The first, qRT-PCR was performed to investigate the expression levels of the 23 AT-hook motif in the life cycle of M. oryzae. As a result, we selected 6 genes(MoATH08, MoATH11, MoATH13, MoATH07, MoATH17, MoATH22) among 23 AT-hook motif genes. A deletion mutant for MoATh08, MoATH07 were made via homology-dependent replacement method. The ΔMoATh08 mutant showed slightly reduced mycelial growth, conidiation, germination rate. However ΔMoATh08 showed a remarkable reduction in pathogenicity by hyphae and conidia inoculation. These data indicate that ΔMoATh08
play a role in cell development and pathogenic development in Magnaporthe oryzae.
Also, functional analysis of the other ΔMoATh07 mutant are in progress.

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 요 약 문 ... 3
• SUMMARY ... 6
• CONTENTS ... 7
• 목 차 ... 8
• 제 1 장 연구개발과제의 개요 ... 10
• 제 1 절 연구개발의 목적과 필요성 ... 10
• 1. 연구개발의 목적 ... 10
• 2. 연구 개발의 필요성 ... 12
• 제 2절 연구개발의 내용 및 범위 ... 14
• 1. 최종연구목표 ... 14
• 2. 연구개발의 내용 ... 14
• 제 2 장 국내외 연구개발 현황 ... 15
• 제 1 절 국내 연구 현황 ... 15
• 제 2 절 국외 연구 현황 ... 15
• 제 3 장 연구개발수행 내용 및 결과 ... 16
• 제 1 절 상세분석을 위한 유전자 선발 ... 16
• 1. Bioinformatics tools을 이용한 유전자 선발 ... 16
• 2. qRT-PCR을 이용한 유전자 발현 조사 ... 21
• 제 2 절 유전자결실돌연변이체 생성 ... 28
• 1. 벼도열병균 KJ201의 원형질체 제작 ... 28
• 2. 결실돌연변이체를 생성하기 위한 construct 제작 ... 29
• 3. homology-dependent gene replacement 방법에 의한 형질전환 ... 30
• 4. 결실돌연변이체 screening ... 33
• 제 3 절 유전자복원 형질전환체 생성 ... 45
• 제 4 절 유전자의 기능 분석 ... 45
• 1. MoATh08의 기능 분석 ... 45
• 2. MoATh07의 기능 분석 ... 49
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 51
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 52
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 53
• 제 7 장 연구시설‧장비 현황 ... 54
• 제 8 장 참고문헌 ... 55
• 연구개발보고서 초록 ... 56
• 끝페이지 ... 57