초록 ▼

1단계 연구의 목표는 야생종 O. minuta (BBCC)의 DNA microarray와 EST 분석을 통하여 내병충성 (상처, 도열병, 멸구에 대한) 유전자들을 대량 선별하고, 내병충성 관련 조절유전자 전장을 클로닝하여 기초적 기능을 분석하는데 있다.
1. 야생벼 EST database
야생벼 O. minuta를 4주 동안 키운 잎에서 추출한 mRNA로 제작된 cDNA library로부터 5,211개 clone들의 염기서열을 분석하여 GenBank dbEST에 등록을 하였고, Horthern blot 분석을 통해 7개

1단계 연구의 목표는 야생종 O. minuta (BBCC)의 DNA microarray와 EST 분석을 통하여 내병충성 (상처, 도열병, 멸구에 대한) 유전자들을 대량 선별하고, 내병충성 관련 조절유전자 전장을 클로닝하여 기초적 기능을 분석하는데 있다.
1. 야생벼 EST database
야생벼 O. minuta를 4주 동안 키운 잎에서 추출한 mRNA로 제작된 cDNA library로부터 5,211개 clone들의 염기서열을 분석하여 GenBank dbEST에 등록을 하였고, Horthern blot 분석을 통해 7개 유전자를 선별하였음.
2. 야생벼 DNA microarray database
세 가지 종류의 subtracted library를 구축하고 1,138 clone들에 대하여 microarray를 실시하였으며, 이를 근거로 8 개의 유용 유전자를 선별하였음 (도열병 처리-omfi; 멸구 처리-omii; 상처 처리-omwi).
3. 선별된 야생벼 내병충성 관련 유전자 기능연구 및 promoter 분석
선별된 15 개의 유전자에 대한 기능분석을 위하여 각 유전자에 대한 전장과 일부 유전자의 promoter 부위를 확보하였으며, 재배벼와의 발현 양상을 비교하고, 형질전환체 및 yeast two hybrid library를 구축하였음.
4. Arabidopsis secretory phospholipase $A_2\;(sPLA_2)$ 유전자의 기능 분석
4 개의 Arabidopsis $sPLA_2$, isoform들을 확보하여 enzymaic activity 등을 측정하여 기능을 분석하였음.

Abstract ▼

Objectives
Wild relatives of the Oryza genus have been regarded as important sources of useful genes for rice breeding, because of their resistance to diseases and pests, tolerance for abiotic stresses. Comparative analysis of gene expressions between cultivated and wild rice can be used to promo

Objectives
Wild relatives of the Oryza genus have been regarded as important sources of useful genes for rice breeding, because of their resistance to diseases and pests, tolerance for abiotic stresses. Comparative analysis of gene expressions between cultivated and wild rice can be used to promote high-throughput screening and bulk gene mining from wild rice efficiently. Moreover, these novel resistant gene collections will not only offer materials for molecular breeding but also serve as catalysts for developing more detailed and principal understandings of the basal mechanisms of plant defense systems. In this project, to isolate and characterize wild rice-originated useful genes, Oryza minuta (BBCC, 2n = 48) was used for PCR-based subtraction, microarray, EST analyses.
Background
The importance of wild relatives has been emphasized in the context of potential genetic sources of resistance to various stresses. Intensive crop breeding narrows genetic diversity in cultivated species by concentrating the many favorable alleles that existed in early domesticates. The wild relatives of rice have diversified in a wide range of environments over a period of 40 million years, and could potentially provide various economic characteristics not easily available in the cultivated germplasms. The value of wild rice have been emphaSized and studied as a resistance gene source. In spite of its overall inferior appearance, O. rufipogon (AA genome) is known to contain genes for salinity tolerance and for the cytoplasmic male sterility, and it also contains useful QTL alleles for certain agronomically important traits. It was also reported o. officinalis (CC genome) resists vermin, such as yellow stern borer, planthopper and leafhopper. These studies suggest that the wild germplasms may offer solutions for future increases in rice productivity, which are also possibly applicable to other crop species. o. minuta, a wild species of rice, has the BBCC genome, which has been used as a donor of resistance to blast and bacterial blight. Due to its important agronomic traits, this species is of prime importance to rice breeders.
Construction of subtracted libraries
Three kinds of subtracted libraries (omfi, rice blast-infected; omii, planthopper-infested; omwi, wound-treated) were constructed from biotic and abiotic stress-treated O. minuta through the combination of suppression subtractive hybridization and mirror orientation selection, the modified subtraction method. To discriminate the differentially expressed transcripts from the background molecules, micro array was carried out as a reverse northern blot in triplicate, and a total of 1,138 clones were selected through data sorting with signal intensity and expression ratios. EST produced from the three kinds of subtracted libraries showed 68.1% of redundancy, and 46.6% of the total clones were matched to the GenBank database. Functional categorization showed that categories of subcellular localization, metabolism, and protein fate were enriched in subtracted libraries.
Statistical analysis and expression profile
To discriminate the artifacts from the microarray data and control the data quality among the technical replications, ANOVA was applied to the second micro array data. From the proposed model for data analysis, the most essential variable in data Variation was the dye effect, while the overall experimental conditions among the technical replications were homogeneous. The effects of the variables on the estimates were validated, proving that our experimental strategies, which were the two dyes system used simultaneously, the experimental replications, and the investigation of interactions between dye and replication, were well adapted for controlling the various artifacts.
By hierarchical clustering of those partial cDNA clones that showed increased expression in the various treatment conditions (time series of fungal infection, insect infestation, and wound treatment), seven clusters (included 305 kinds of partial transcripts) were generated. Eighty transcripts displayed elevated expression in response to the three different kinds of stresses, and one hundred sixty-three transcripts were classified in the hormone-induced group. Gene expression levels were further confirmed by Northern blot analysis.
Comparative expression and functional characterization of the selected clones
Based on the hierarchical analysis, 25 clones were chosen and validated their expression using Northern blot analysis, and 17 clones showed similar patterns on both analyses. From the micro array, Northern blot, and EST analyses, the candidate clones were selected for functional analyses. Their comparative Northern blot analysis using stress-treated O. sativa and O. minuta showed the differential expression pattern, suggesting that the defense mechanism of wild rice is possibly derived from not only wild rice specific novel gene but also the regulation of gene expression. Subcellular localization of two selected clones were confirmed using the smGFP fusion vector, and these results revealed that the clones contain the mitochondria- targeting sequence information.
Construction of EST database from a normal leaf cDNA library
A cDNA library was constructed from 4-week-old leaf samples of greenhouse grown O. minuta. The 5,211 cDNA clones of O. minuta represented a total of 3,401 unique sequences, which consisted of 2,787 singletons and 614 assembled sequences. Database comparisons of the cDNAs in GenBank's non-redundant databases using BLAST revealed that 4,957 of the 5,211 cDNAs (95.1%) showed a high degree of sequence homology to genes from other organisms. Through the MIPS functional categorization, we found that most of the identified transcripts were classified to metabolism, energy, protein biosynthesis and subcellular localization. The metabolism and energy categories of O. minuta ESTs showed a considerably higher gene expression level than those of O. sativa.
Expression patterns of transcription factors selected from a normal EST library
Based on DB search, EST clones related to signal molecules to biotic and abiotic stresses were chosen. After confirming their expression patterns using Northern blot analysis, seven clones were selectedi four transcription factors, two kinases and one novel gene. All the selected clones existed as a single or low copy not only in o. minuta genome but also in those of O. sativa. To verify the expression pattern of the selected ESTs between o. minuta and O. sativa, comparative Northern blot analyses were used. In this comparison, we could observe the differential expressions under the fungal, insect, and wound treatments. Five orthologs from O. sativa were identified for the comparative functional analysis. To characterize their functions, full-length cDNAs were cloned through the RACE teclmique, and transfonned to O. sativa and Arabidopsis.
Summary for the microarry and EST data
To identify the differentially expressed transcripts in O. minuta by fungal infection, insect infestation and wound treatment, both EST screening and SSH combined with micro array were used to eliminate false positive clones and to increase selection efficiency. Based on expression profiling, Northern and Southern blot analysis, 8 clones were selected for the functional study. All tested clones existed as a single or low copy on genomes of O. sativa as well as O. minuta. From the 5,211 normal ESTs of O. minuta, 5 transcription factors, 2 kinases and one novel gene, which possibly related to biotic and abiotic stresses, were selected for their functional study.
Functional analyses of Arabidopsis secretory phospholipase A₂(sPLA₂) genes isolated from our previous screening
To elucidate the cellular functions of phospholipase A2 in plants, Arabidopsis cDNAs encoding secretory phospholipases A₂ (AtsPLA₂-β, -γ, and -δ) as well as AtsPLA₂-α were isolated in our previous massive screening. Quantitative RT-PCR analyses revealed that AtsPLA₂s mRNA showed dominant expression pattern in floral organs, especially AtsPLA₂-δ exhibited the organ specific expression in flower. In onion epidermal cells, AtsPLA₂-β, -γ, and -δ were secreted into the intercellular space, consistent with the existence of their signal peptide. Kinetic analyses indicated that AtsPLA₂-β, -γ and -δ have a sn-2 position specific phospholipase activity and a head group preference (Lee et al., 2003 and Bahn et al., 2003).
In the analyses of promoter::GUS transgenic plants, AtsPLA₂-β transcripts revealed prominent in the overall tissues with tissue- and stage-specific manner, whereas the expression of AtsPLA₂-γ and -δ were restricted in pollen and anther sac. The overexpression of AtsPLA₂-β promoted cell elongation, whereas RNA interference-mediated Silencing of AtsPLA₂-β retarded cell elongation. As results, AtsPLA₂-β-overexpressed or -silenced transgenic plants revealed that the altered gravitropism in inflorescence stems and hypocotyls possibly is related with auxin signaling (Lee et al., 2003). On the other hand, RNA interference-mediated silencing of AtsPLA₂-γ and -δ expression retards the formation of lipid droplet on pollen coat and subsequently inhibited pollen germination.
Further research plan
Fifteen O. minuta genes and their O. sativa and Arabidopsis orthologs have been selected in our previous researches using micro array and EST analyses. To characterize their biological functions, several functional genomic approaches will be applied during three years' second stage. Following approaches are planed: (1) Yeast two-hybrids to identify the interacting protein genes, (2) Promoter anayses and yeast one-hybrids to identify upstream regulatory genes, (3) Transformation of those genes into O. sativa and Arabidopsis, (4) Knock-out mutants and overexpressed mutant studies, (5) Analyses of genomic structure and alternative splicing patterns.
In this project, we will can elucidate gene effects and components of regulatory networks that mediate defense signaling, and finally can provide some valuable genes for crop breeding program toward the enhanced resistance to various stresses.

목차 Contents

• 표지...1
• 제출문...2
• 보고서 초록...3
• 요약문...4
• SUMMARY...9
• CONTENTS...14
• 목차...15
• 제 1 장 연구개발과제의 개요...16
• 제 2 장 국내외 기술개발 현황...19
• 제 3 장 연구개발 수행 내용 및 결과...21
• 제 4 장 목표달성도 및 관련분야에의 기여도...44
• 제 5 장 연구개발결과의 활용계획...46
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보...48
• 제 7 장 참고문헌...49