보고서 정보
주관연구기관 |
국립농업과학원 |
연구책임자 |
김동헌
|
참여연구자 |
윤웅한
,
김둘이
,
윤인선
,
김창국
,
서미숙
|
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2012-01 |
과제시작연도 |
2011 |
주관부처 |
농촌진흥청 |
사업 관리 기관 |
농촌진흥청 Rural Development Administration |
등록번호 |
TRKO201200009809 |
과제고유번호 |
1395021656 |
DB 구축일자 |
2013-05-20
|
초록
○ 벼 성장관련 transcription factor 발굴 및 유전자 발현 분석
- 종자 발달, 분얼 등 관련 유전자 발현 마이크로 어레이 분석
○ 벼, 배추 유전자발현 마이크로어레이 데이터 분석 시스템 구축
○ 벼 MG RIL 164 계통의 광합성 특성 및 메타볼롬 프로파일 분석
○ 배추 glucosinolate 생산 조절 transcription factor 발굴 및 유전자 발현 분석
Abstract
▼
Many research groups try to build system on the entire rice genome information since completed rice genome sequence at the International Rice Genome Sequencing Project (IRGSP) in 2005. In this research, we studied the expression profiling and the functional analysis of transcription factor in rice i
Many research groups try to build system on the entire rice genome information since completed rice genome sequence at the International Rice Genome Sequencing Project (IRGSP) in 2005. In this research, we studied the expression profiling and the functional analysis of transcription factor in rice ilpumbyeo. 73,000 genes were sequenced in rice seed of ilpumbyeo including 368 transcription factor genes. We determined full-length cDNA sequences of the 193 transcription factors. In the functional analysis of the rice transcription factor, we selected a variety of insertional mutant lines, and field-test was carried out to analyze the characteristic function of transcription factor genes. The 32 lines exhibited interesting phenotype with regard to seed size, pigment variation, heading, and opaque form. Zinc finger insertion mutants, 1A-09223 showed a smaller seed size than wild-type, and b-zip mutant, 1A-14632 were found exhibiting shattering habit as well as smaller grain. RNA was extracted for 135K microarray experiments to investigate morphological variation lines such as b-zip mutant. Based on the gene expression profiles of 135K microarray results, it was that 2,590 genes expressed tissue-specifically, and 22 genes among 72 heat shock proteins in the seeds were isolated. We analyzed the transcription factors involved in seed-morphogenesis such as 104 Myb genes and 89 NAC genes. Moreover, 12 seed specific transcription factors were isolated. Our microarray results were arranged in NABIC database (National Agricultural Biotechnology Information Center) and opened to the public. Our data provide comprehensive information for researchers studying rice functional genomics.
LIM proteins play key roles in a variety of important development processes. They all contain one or several double zinc finger motifs of sequence and the domains, which functions by mediating protein-protein interactions. Also, associated with a DNA-binding homeodomain, they operate as transcription factors. The coding region of a rice LIM-domain protein genes were obtained by RT-PCR from early seed development stage. A OsLIM gene was contains an open reading frame of 675bp, coding for a polypeptide of 225 amino acids with an estimated molecular 24.8 kDa. Also, a OsPLIM gene contain domain similar to OsLIM gene was identified an open reading frame of 621bp, coding for a polypeptide of 206 amino acids with an estimated molecular 22.5 kDa. We were found a LIM-domain with the conserved sequence of a double zinc-finger structure in OsLIM and OsPLIM genes. We were screened proteins which interacts with OsLIM gene using yeast two-hybrid system. The results of screening, homeobox containing protein, WD40, target of Myb1 like, ring finger, actin binding protein and elongation factor gamma2 genes are selected. To functional analysis of OsLIM and OsPLIM genes, we are identified insertion to OsLIM and OsPLIM genes in rice transgenic plants. We were phenotype analysis to 17 lines OsLIM and 50 lines OsPLIM transgenic plants. Among their transgenic lines, the OsLIM and OsPLIM lines have been characterized to decrease tiller numbers and bigger seed size than wild plant. We are investigating to functional characteristic of LIM-domain using transgenic plant.
Shoot branching is a major determinant of plant architecture and therefore, an important agronomic trait for grain yield. Shoot branches arise from the axillary meristem, whose activity is regulated by the interplay of genetic programs with the environment. Recent results showed that these signals are integrated through interacting hormonal and transcription factor regulatory networks. In order to identify key regulators involved in the shoot branching process of rice, we have investigated transcriptome, proteome and metabolome profiling of a rice high tillering and dwarf (HTD) mutant. We have identified 27 metabolic pathways, 198 transcription factors, 60 proteomes and 69 NMR peaks in the HTD mutant. These provide valuable information for controlling rice tiller formation. TCP (Teosinte branched/Cycloidea/PCNA) family was selected as highly enriched transcription factors in the merisetmic region of the HTD mutant. The TCP family is known as important regulators of plant cell division and organ development. Molecular functions of OsTCP6 and OsTCP11 were further investigated. These proteins were characterized as nuclear localized transcriptional activators. Ectopic expression of OsTCP6 or OsTCP11 in Arabidopsis modified leaf growth and development, and genes related to cell expansion and cell cycle were found to be up-regulated. In transgenic rice over-expressing OsTCP6 and OsTCP11, tillering was activated at earlier stages. Our present data suggest that OsTCP6 and OsTCP11 are positive players of lateral organ growth and development.
The microarray-based database has 5,000 records which gene expression information for agricultural plants. It provides a bioinformatics framework to study biological function based on microarray gene expression of rice and Chinese cabbage. It provides useful information through a user-friendly web interface that allows analysis of gene expression pattern and searching of various resources. Especially, to examine gene expression involved in anthocyanin biosynthesis in Chinese cabbage (Brassica rapa), we performed computational analyses using a 300K Brassica rapa microarray. In addition, to better understand the functional characterization of anthocyanin gene expression in black rice, we performed a detailed computational examination. The experiment was performed using the designed 135K Oryza sativa microarray to assess gene expression in Ac/Ds insertion lines.
By entering the post-genome era, it becomes more important to determine relationships of genes in expression in order to understand the roles of genes in a genome. To achieve the goal, the microarray technique is one of the important methods to identify expression patterns and interactions between genes on the genomic scale. As a result, the gene expression data from microarray analysis have been increased exponentially. However, Bias occurs frequently by systematic variation in microarray experiments. Thus, in order to determine general relation between genes, the normalization process is essential to adjust the systematic variation. However, to deal with the many microarray data sets is complex and troublesome and is especially impossible for many biological researchers who are unfamiliar with computational work. Thus, we built a database, named PlantArrayNet (PAN) in order to determine relations between genes in accumulated gene expression profiles built in genomic scale. The PAN provides correlating infor mation between genes through relational tree, network and hierarchical cluster in gene expression based on the correlation levels between genes evaluated by Pearson’s correlation coefficient. Additionally,the PAN provides scatter plot of log ratio between genes, related pathway maps and cis-element list of promoter regions. Currently, the PAN provides correlating information of three plant organisms; Oryza sativa, Arabidopsis thaliana and Brassica rapa. The PAN is useful tool to understand expressional relation between genes in order to understand the roles of genes in a genome. The PAN is freely accessible at http://bioinfo.mju.ac.kr/arraynet/.
In this study, photosynthetic activities of 164 Inbred lines obtained by crossing between Milyang23 and Giho-byeo were measured. Although the photosynthetic carbon assimilation rates of parental lines, Milyang23 and Giho-byeo, were not much different at 400μ mol·mol-1,CO2 and 1000μmol·m-2 PAR, there is a large variation in photosynthetic carbon assimilation rates among the population of MG RILs. Several lines were selected based on their photosynthetic carbon assimilation rates and used as a material for the analysis of enzyme activity involved in Calvin cycle. Measurement of the activities of enzymes revealed that RubisCO initial and total activities are to some extent related to the variation of photosynthetic carbon assimilation of MG RIL population, while others such as FBPase are not directly related. Based on this information, RubisCO activities were measured using whole population of MG RILs. Results showed that RubisCO initial activities as well as total activities were varied although majority of the MG RILs showed limited RubisCO acitivities. QTL analysis showed that there are one and two QTL loci related to the carboxylation efficiency of MG RIL' s photosynthetic carbon assimilation activities.In this research time course changes of photosynthetic activity and leaf conductance upon salt treatment were also monitored. Such changes appeared to be related to the response to salt treatment of plants examined. The protocol developed in this experiment could be useful for the phenotypic analysis of transgenic plants developed for abiotic stress resistance.
Improvement of crop species has been a fundamental human pursuit since cultivation began. In face with the climate change, which could cause irreversible or reversible damage to agricultural echo system and loss of crop productivity, the development of crop species will contribute significantly to crop development. Plant responses to abiotic stresses are dynamic and complex, and they involve interactions and crosstalk with many molecular pathways. For the development of crop species, the understanding and fine regulation of regulatory network is required. Systems biology and omics approaches can be used to elucidate the key regulation pathways to genetic or environment factors in plants. Metabolomics approaches enable the parallel assessment of the levels of a broad range of metabolites and have great value in both phenotyping and diagnostic analyses in plants. These tools also can be used to evaluate the natural variance apparent in metabolite composition.
We applied metabolmics approach to characterize the genetic diversity of rice. The 1H-NMR based untargeted metabolic profiling with combination of multivariate statistical analysis cannot discriminate efficiently the photosynthesis efficiency based genetic variants,which may caused by the diversity of metatabolic phenotypes is overwhelming the differences of photosynthesis. Target analysis of photosynthetic intermediates showed that the differences of theses metabolites were trivial. The discrimination of salt stress responses in salt sensitive and resistant rice cultivar roots was apparent depending on salts responsiveness. In salt sensitive cultivar, Nakdong, the major salt responsive factors were lysophosphatidycholine and phosphatidylcholine, which were known as signal molecules responding to various stresses in plants and animals. Some kinds of lipid amide were identified as increasing factors in roots of salt resistant rice cultvar, Pokkari, and soluble auxin binding protein ABP57 over-expressing transg enic rice roots. Many of the lipid amides were reported as displaying potential biological activities at low concentrations. Contrary to roots, leaf metabolite patterns were not changed by salts treatment in Pokkari. Tissue dependency of stress response is well known. Though the function of major metabolic factors responding to salts stress in this study need validation, our results show that metabolomics approach can be a powerful tools for characterization and understanding of environmental stress response regulatory network.
Glucosinolates(GSLs) are secondary metabolites synthesized from several amino acids in the Brassicaceae family. Methionin-derived aliphatic glucosinolates have recently attracted attention because its anticarcinogenic activity. We identified genes related to aliphatic glucosinolate biosynthesis of Brassica rapa by comparative genome analysis with Arabidopsis. The results were suggested that the glucosinolate regulation of B. rapa have more complex pathway than the Arabidopsis because complexity of B. rapa genome following polyploidy. The three members of MYB28 transcription factor and two genes (AOP2, GSL-OH) related to side-chain modifications in the aliphatic glucosinolate pathway were isolated. These genes transferred to B. rapa by overexpression and gene silencing using Agrobacterium for funtional analysis. We confirmed by PCR that all hygromycin-resistant plants retained stable insertion of the hpt gene. The results of RT-PCR also demonstrated that the variations in expression levels of the genes in some transformants were caused by gene insertion. We are analyzed the variations of glucosinolate levels in leaves of transgenic plants by HPLC. The results resulted in the increase of aliphatic glucsinolates and total glucosinolate content in transgenic plants. The results also showed that gene silencing of the BrGSL-OH gene family resulted in dramatic increase in contents of glucobrassicanapin or sinigrin in transgenic plants. We are currently selecting the T2 homozygous transgenic lines for gene expression network analysis related to glucosinolate biosynthesis in B.rapa.
목차 Contents
- 표지 ... 1
- 제출문 ... 2
- 요약문 ... 3
- SUMMARY ... 4
- 제1장 서 론 ... 9
- 제1절 농업형질관련 전사조절 인자 발현 profiling 분석 및 기능해석 ... 9
- 제2절 종자형성 관련 유전자의 최적 발현 조건 확립에 의한 분자육종 소재 개발 ... 9
- 제3절 벼의 분얼을 조절하는 전사인자 네트워크 연구 ... 10
- 제4절 벼, 배추 유전자 발현분석 데이터 DB 구축 ... 11
- 제5절 벼, 배추 발현 네트워크 DB 구축 및 비교전사체 연구 ... 11
- 제6절 벼 유전집단 광합성 특성 분석 및 대사조절 유전자 발굴 ... 12
- 제7절 대사체 분석을 통한 벼 유전자원 특성 연구 ... 13
- 제8절 배추 글루코시놀레이트 합성관련 유전자 구조분석 및 발현조절 ... 14
- 제2장 국내외 기술개발 현황 ... 16
- 제1절 농업형질관련 전사조절 인자 발현 profiling 분석 및 기능해석 ... 16
- 제2절 종자형성 관련 유전자의 최적 발현 조건 확립에 의한 분자육종 소재 개발 ... 16
- 제3절 벼의 분얼을 조절하는 전사인자 네트워크 연구 ... 17
- 제4절 벼, 배추 유전자 발현분석 데이터 DB 구축 ... 17
- 제5절 벼, 배추 발현 네트워크 DB 구축 및 비교전사체 연구 ... 18
- 제6절 벼 유전집단 광합성 특성 분석 및 대사조절 유전자 발굴 ... 18
- 제7절 대사체 분석을 통한 벼 유전자원 특성 연구 ... 21
- 제8절 배추 글루코시놀레이트 합성관련 유전자 구조분석 및 발현조절 ... 22
- 제3장 연구개발수행 내용 및 결과 ... 24
- 제1절 농업형질관련 전사조절 인자 발현 profiling 분석 및 기능해석 ... 24
- 제2절 종자형성 관련 유전자의 최적 발현 조건 확립에 의한 분자육종 소재 개발 ... 48
- 제3절 벼의 분얼을 조절하는 전사인자 네트워크 연구 ... 59
- 제4절 벼, 배추 유전자 발현분석 데이터 DB 구축 ... 66
- 제5절 벼, 배추 발현 네트워크 DB 구축 및 비교전사체 연구 ... 71
- 제6절 벼 유전집단 광합성 특성 분석 및 대사조절 유전자 발굴 ... 75
- 제7절 대사체 분석을 통한 벼 유전자원 특성 연구 ... 91
- 제8절 배추 글루코시놀레이트 합성관련 유전자 구조분석 및 발현조절 ... 103
- 제4장 연구개발목표 달성도 및 대외기여도 ... 112
- 제1절 목표대비 달성도 ... 112
- 제2절 정량적 성과 ... 114
- 제5장 연구개발결과의 활용계획 ... 121
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