초록 ▼

○ 연구결과
1) 도열병에 의해서 유도된 유전자 1200여종을 DD, SAGE, DSC 방법으로 screening하여 microarray로 확인해서 서로 다른 16종류의 유전자를 확인 했다. 2-DE을 통해서 방어관련 단백질을 14종류 확인해서 그중 4종류 (PBZ1, OsPR-10, OsIRL, SalT)의 antibody을 생산 그 기능 연구에 사용했으며, 그 중 PBZ1의 기능이 cell death와 관련이 있다고 결과를 얻었다.
2) NIM1-interacting protein 으로서 2 종의 bZIP protei

○ 연구결과
1) 도열병에 의해서 유도된 유전자 1200여종을 DD, SAGE, DSC 방법으로 screening하여 microarray로 확인해서 서로 다른 16종류의 유전자를 확인 했다. 2-DE을 통해서 방어관련 단백질을 14종류 확인해서 그중 4종류 (PBZ1, OsPR-10, OsIRL, SalT)의 antibody을 생산 그 기능 연구에 사용했으며, 그 중 PBZ1의 기능이 cell death와 관련이 있다고 결과를 얻었다.
2) NIM1-interacting protein 으로서 2 종의 bZIP protein을 분리 하였고, 천연적으로 아미노 말단 부위가 일부 제거된 7종의 bZIP factor를 얻게 되었도 이들의 결합부위 규명, 프로모터 인식규명, 발현 분석으로 형질 전환체에서 광범위 병저항성 개발의 기초를 놓았다.
3)constitutive expression cassette T-DNA vector 구축 및 이를 통한 형질전환체 개발 원하는 유전자가 ectopic 그리고 constitutive expression되는 형질전환체를 만들기 위한 T-DNA vector 구축되었고 wounding에 반응하는 유전자인 OsSNF1 cDNA를 삽입하여 형질전환체를 생산하였다. wounding 에 반응하는 OsSNF1을 동정하고 promoter를 분리하여 GFP/GUS가 wounding과 도관에 특이적으로 발현하는 형질전환체를 생산하였다.
4) 병저항성 유전자 R genes의 ‘knock-out' 선발/육성
1,000 개 이상의 Ds flanking DNA의 염기서열 분석을 통하여 Ds가 R 유전자에 들어가 유도된 knock-out 변이 선발하고 벼 저항성 연구에 사용될 유전 자료 확보

Abstract ▼

To identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea, we have devised and exploited differential screening techniques such as DSC, and differential hybridization and microarray. We isolated 768 genes and determined expression levels of their g

To identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea, we have devised and exploited differential screening techniques such as DSC, and differential hybridization and microarray. We isolated 768 genes and determined expression levels of their genes. Among these genes, β-1,3-glucanase, PBZ, PR-10, SaIT(defense-stressresponses), sucrose-UDP glucosyltransferase, L-allo-threonine aldolase(metabolism), OsGAI (hormonal response), and mitogen activated protein kinase(MAPK) (signal transduction) gene expression levels were increased after blast inoculation. Northern blot analysis also confirmed that the expression of these genes were induced by treatment of fungal elicitor.
Serial analysis of gene expression(SAGE) offers the cataloging of the identity and relative frequencies of mRNA transcripts. Through SAGE analysis, we screened differential transcripts of incompatible and compatible interaction between rice - rice blast fungus. Total of 2,745 tags were isolated, 910(33%) tags were identified but 1,850(67%) could not be identified from GenBank database at this moment. Among identified tags, 13.5% , 4.1% tags showed stress/pathogen and signal transduction related gene, respectively.
We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H2O2. The proteins were then PEG fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier an to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.
The temporal expression of a probenazole-induced protein(PBZ) was paralleled with several developmental events associated with programmed cell death (PCD) occurred in leaf senescense, root aerenchyma formation, coleoptile, root cap, seed aleurone layer, and hypersensitive-response. The expression levels and localizations of PBZ in tissue of PCD were identified by Western blot and immunohistochemistry analysis. Purified recombinant PBZ was also induced hypersensitive cell death in rice suspension cells and tobacco leaves in dosage dependent manner. Pretreatment of antibody raised against PBZ blocked cell death caused by PBZ. These results demonstrated that PBZ was involved in plant cell death.
NIM1/NPR1 is a critical component of the salicylic acid (SA)-mediated signal transduction pathway. From fugal infected rice leaf tissues, we have isolated two rice NIM1 interacting proteins (OsNIPs) using a well defined screening system, yeast two-hybrid screen. These two OsNIPs are members of the TGA family of basic leucine zipper (bZIP) transcription factor. As reported elsewhere, the amino-termini of these factors were found to decrease binding stability in yeast and differentially affect their apparent affinity toward NIM1. The effect of NIM1 binding to OsNIPs was studied in view of protein-DNA interaction. The presence of NIM1 enhanced binding of OsNIP1 to as-1 and nos-1, cis-elements containing TGACG-motifs found in PR promotors.
The rapid induction of OsNIP1 messages by elicitor and jasmonic acid is interesting in view of the fact that NIM1 gene was not induced by salicylic acid and other defence signals. This raise the possibility that the rapid induction of OsNIPs is a first event that occur after elicitor treatment, which is required for PR gene expression. The role of NIM1-OsNIP interaction on gene expression will require further investigation.
Development of inducible promoter and construction of T-DNA vectors for constitutive and inducible expression of genes related to pathogen resistance and selection and breeding of knock-outs of disease resistance genes (R) via Ds insertion were carried out to develop transgenic plants. . To achieve the goals, we identified a rice gene, OsSNF1, that is wounding responsive and vascular tissue-specific. Using the promoter of this gene, wounding-inducible expression cassette of T-DNA vector was constructed.
Rice genome has around 600 NB-ARC homologous genes that might be related to pathogen resistance in one way or another. Therefore, understanding the functions of these genes is very important to molecularly dissect pathogen-resistance in rice. After characterizing more than 1,000 Ds loci, we found 12 NB-ARC genes and established them as stable lines. For future study, these lines will be important materials in analyzing pathogen-resistance.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 6
• CONTENTS ... 9
• 목차 ... 10
• 제1장 연구개발과제의 개요 ... 11
• 1절 연구개발의 필요성 ... 11
• 2절 추진전략 ... 15
• 3절 연구방법 ... 16
• 제2장 국내외 기술 개발 현황 ... 24
• 제3장 연구 내용 및 결과 ... 25
• 1절 벼 도열병 저항성 관련 유전자 cloning 및 특성연구 ... 25
• 2절 생체방어 관련 전사조절인자인 rice NIM1/NPR1 interacting protein 분리 및 기능연구 ... 68
• 3절 식물생체 방어 관련 유전자의 형질전환에 적합한 promotor의 개발 및 형질 전환체 개발 ... 81
• 제4장 목표달성도 및 관련 분야에의 기여도 ... 95
• 제5장 연구개발결과의 활용계획 ... 98
• 1절 기대효과 ... 98
• 2절 활용방안 ... 99
• 제6장 연구개발과정에서 수집한 해외 과학기술의 정보 ... 101
• 제7장 참고문헌 ... 102
• 논문발표 ... 105
• 끝페이지 ... 106