초록 ▼

Ⅳ. 연구개발결과
ⅰ. 애기장대 ICU2에 의한 메틸화 히스톤의 전달과정 연구
유전적으로 연관성이 있을 것으로 보이는 CLF와 및 homolog인 MEA, SWN과 ICU2의 상호작용을 yeast two hybrid와 애기장대 protoplast에서의 BiFC를 통해 확인한 결과, 서로 N-terminal domain을 통해 상호작용함을 확인하였다.

ⅱ. 애기장대 DME에 의한 DNA 메틸화 패턴의 전달과정 연구
수배우체의 발달과정에서 DME 유전자가 bi-cellular stage의 vegetative

Ⅳ. 연구개발결과
ⅰ. 애기장대 ICU2에 의한 메틸화 히스톤의 전달과정 연구
유전적으로 연관성이 있을 것으로 보이는 CLF와 및 homolog인 MEA, SWN과 ICU2의 상호작용을 yeast two hybrid와 애기장대 protoplast에서의 BiFC를 통해 확인한 결과, 서로 N-terminal domain을 통해 상호작용함을 확인하였다.

ⅱ. 애기장대 DME에 의한 DNA 메틸화 패턴의 전달과정 연구
수배우체의 발달과정에서 DME 유전자가 bi-cellular stage의 vegetative cell 에서만 발현하는 것을 새로이 밝히고, DME 유전자 발현 조절인자는 Sporophytic tissues와 central cell, vegetative cell에서 각기 다르며 개별적으로 작용함을 확인하였다.

ⅲ. 벼 DNA demethylase 벼 생식기관 발달에서 작용기작 분석
벼의 DNA 탈메틸화에 관여하는 Ros1b,1c, 1d와 Dml3b의 기능상실, Ros1의 기능활성 돌연변이를 확보하고, 이중 순종 분리 된 Ros1c의 기능상실 및 Ros1b, 1c의 기능활성 돌연변이의 발현 및 표현형 확인하였다. 또한, 각 유전자의 Ros1a, 1b, 1c, 1d promoter-GUS 형질전환체 제작하여 Ros1a, 1b, 1c, 1d promoter-GUS, Ros1a promoter-GUS 형질전환체에서의 각 유전자 발현위치 및 시기 확인하였다.

ⅳ.벼 DNA methylase 작용 메커니즘 분석
OsMet1 기능상실 돌연변이체 1종 분리을 분리하였고, OsMet1 발현조직 확인하기 위해 6종의 construct에 대한 형질전환체 확보하여 OsMet1의 세포내 위치 확인, osmet1-1 5종 유전자에 대한 methylome 분석하였다.

Abstract ▼

Gene expression is not only altered by the genome structure but also by epigenetical causes. Epigenetical markers have been observed at all stages of plant growth and development including seed development. In particular, epigenetical gene expression alteration during the double fertilization gives

Gene expression is not only altered by the genome structure but also by epigenetical causes. Epigenetical markers have been observed at all stages of plant growth and development including seed development. In particular, epigenetical gene expression alteration during the double fertilization gives rise to apomixis and seed quality changes in Arabidopsis thaliana and Oriza sativa.
The methylation of DNA and histone proteins are known as the main markers of epigenetical aspects having direct and indirect effects on gene expression. The seed size of mutant Arabidopsis DNA methyltransferase MET1 and mutant DNA demethylase DEMETER (DME) differs from wild-type. Still the function of the homologous genes of DME or MET1 in rice have not been reported yet. Clarification of Arabidopsis DME expression regulation and functional studies with rice mutants of 6 DME family genes, 2 of MET1 homologous genes are performed in this study.
The H3K27me3 (Histone-3-lysine-27-trimethylation) is known as an epigenetical repressor marker. In Arabidopsis, the E(z) component of the polycomb group complex2 (PRC2) works as a methylatransferase, occurring H3K27 methylation, and has importance in flower development. In Arabidopsis INCURVATA2 mutant, H3K27 methylation is not properly inherited between cell divisions and causes an early flowering phenotype. With this mutant, we want to reveal the mechanism of histone modification inheritance.
The Arabidopsis DME is expressed in the central cell, the companion cell of the egg, and the vegetative cell, the companion cell of the sperm during reproduction. DME-mediated DNA-demethylation is required for seed and pollen viability. In addition, DME expression in the shoot and root meristem regions are required for proper sporophyte development. By using a series of iteratively deleted and mutagenized transgenes, combined with T-DNA insertion mutants, we could identify key overlapping enhancer regions necessary for central cell and vegetative cell transcriptional activity, a distinct enhancer for transcriptional activities of DME in the sporophyte. We delineated the pattern of DME expression in pollen, showing that expression is confined to the vegetative cell, and is transient, occurring only during the bicellular stage of development, similar to its transient expression pattern in the ovule.
Rice has two merciless and they are localized in nucleus in common. Homozygous mutants for 2 DNA methylase, OsMet1-1 and OsMet1-2, were identified, and OsMet1-2 appears to be essential thereby osmet1-2/osmet1-2 was not available. To examine the overexpression effects on plant growth and development, transgenic rice plants were generated for the six constructs. Pmet1:: GFP, Pmet1:: Met1: GFP, and Pubi:: Met1: GFP.
Rice has six DME homologous genes ROS1a, ROS1b, ROS1c, ROS1d, DML3a and DML3b. Each gene knockout or overexpressing mutants were isolated for phenotyping. Overexpression of ROS1b produced low fertility. ROS1c knockout or overexpression generated growth problems and a low fertility phenotype. The expression of ROS1a was found in flower lemma and the dorsal area of a developing seed. The ROS1b expression pattern was seed specific and ROS1d was found in the young veins excluding the main vascular tissue, suggesting functions during vascular development. DML3b was expressed in early flower and other genes were globally expressed.
INCURVATA2 (ICU2), the catalytic subunit of Arabidopsis DNA polymerase alpha, Shows early flowering phenotype of icu2-1 missense mutations show evidence of the H3K27me3 level change of the FLC region between cell divisions. In order to find partners of ICU2, a yeast two hybrid screening has been performed. As the results we could find CLF, MEA and SWN, which are components of PRC2 complex interaction with ICU2. They were interacting with ICU2 by their N-terminal domain. ICU2 interacts with those proteins through its N-terminal domain. Bimolecular fluorescence complementation (BiFC) assay was performed and confirmed the interaction with ICU2 in vivo.