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Functional modules of DEMETER 5-methylcytosine DNA glycosylase necessary for seed development in Arabidopsis : 애기장대의 종자발달에 필수적인 DEMETER 메틸시토신 DNA 글리코실라제의 기능성 모듈
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 허진회 | - |
dc.contributor.author | Hyun Jung Rim | - |
dc.date.accessioned | 2017-07-14T06:30:31Z | - |
dc.date.available | 2017-07-14T06:30:31Z | - |
dc.date.issued | 2013-02 | - |
dc.identifier.other | 000000009407 | - |
dc.identifier.uri | https://hdl.handle.net/10371/125650 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 : 식물생산과학부, 2013. 2. 허진회. | - |
dc.description.abstract | DNA methylation is a key epigenetic mark that regulates gene expression. In Arabidopsis, DEMETER (DME) DNA glycosylase specifically removes 5-methylcytosine (5mC) from DNA and induces gene imprinting in endosperm. In heterozygous dme mutants, 50% of seeds are aborted due to a maternal parent-of-origin effect. There are three other DME-like genes such as ROS1, DML2, and DML3 in the Arabidopsis genome, and all DME family proteins share the similar domain structures. Besides the central DNA glycosylase domain, two additional domains (domains A and B) exist and all these are required to excise 5mC. These three domains are interspersed with the interdomain regions (IDRs) which are highly variable in sequence and length among the family members. To identify the minimal fragment necessary for 5mC excision, the DMEΔ(149-677)ΔIDR1::lnk, where the domains are cut and pasted, were engineered. The modified DME fragment still retains biochemical activity. This indicates that the three domains are enough for DME function in vitro.
In the first chapter, DMEΔ(149-677) and DMEΔ(149-677)ΔIDR1::lnk which have DNA glycosylase activity were expressed in the central cell using DME promoter. The modified DME can rescue seed abortion in dme mutants. This demonstrates that the three conserved domains are functional modules which are necessary and sufficient for both in vivo and in vitro functions of DME. In addition, DMEΔ(149-677) could maintain the DME function stably in the next generation. Homozygous dme mutants with lethal phenotype were recovered and abnormal seed and flower phenotype were observed in T2 DMEΔ(149-677)ΔIDR1::lnk population. The results implies that DMEΔ(149-677)ΔIDR1::lnk might be able to have hyperactivity function of DME and that DNA methylation and floral morphology are related in Arabidopsis. In the second chapter, a novel DNA methylome analysis tool using DME was proposed. After DME DNA glycosylase reaction in which DME recognizes and removes mC in Arabidopsis genome, radioactively labeled cytosines were refilled in the vacant position by BER pathway. Here, dot-blot analysis was conducted and DNA methylation pattern was visualized for the specific regions of genome. The result suggests that DME treatment differentiates between hyper- and hypo-methylated genome fragments. If the method would be applied to fluorescent labeling system and tiling array, DME-chip could become the easy and fast DNA methlyome analysis tool through the single tube DME reaction. | - |
dc.description.tableofcontents | ABSTRACTi
CONTENTSiii LIST OF TABLESvii LIST OF FIGURESviii LIST OF ABBREVIATIONSx LITERATURE REVIEWS1 DNA methylation and demethylation in plants 1 Genomic imprinting in angiosperms 3 Principles of genome-wide DNA methylation analysis 9 LITERATURE CITED11 CHAPTER I. Genetic analysis of functional modules of DEMETER methylcytosine DNA glycosylase necessary for seed development in Arabidopsis18 ABSTRACT 18 INTRODUCTION 20 MATERIAL AND METHODS 24 Manipulation of pBIIOI binary vector 24 Molecular cloning of DME fragments 24 Plant materials and growth conditions 28 Agrobacterium mediated Arabidopsis transformation 28 Genomic DNA extraction and genotyping of transgenic lines 29 RNA isolation and gene expression analysis 29 Histological GUS assay 32 Observation of seed phenotype and statistical analysis 32 RESULTS 33 Existence of two DME splicing variants 33 Expression of pDME:DMEΔN:GUS in the transgenic lines 35 DMEΔN and DMEΔNΔIDR::lnk complement dme seed abortion 38 DMEΔN and maintaining of aborted seed rescue in TII generation 44 Unstable seed viability and abnormal seed and flower phenotypes in TII plants with pDME:DMEΔNΔIDR::lnk:HA gene 47 DISCUSSION 50 REFERENCES 54 CHAPTER II. DNA methylation profiling by functional modules of DEMETER in Arabidopsis 57 ABSTRACT 57 INTRODUCTION 58 MATERIALS AND METHODS 60 Plant materials 60 Preparation of sample with Arabidopsis gDNA 60 DME reaction condition and probe preparation 61 Dot-blot assay 62 RESULTS AND DISCUSSION 63 REFERENCES 69 ABSTRACT IN KOREAN 70 | - |
dc.format | application/pdf | - |
dc.format.extent | 1954097 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | DEMETER | - |
dc.subject | DNA demethylation | - |
dc.subject | seed development | - |
dc.subject | functional modules | - |
dc.subject | DNA methylome | - |
dc.subject | Arabidopsis | - |
dc.subject.ddc | 635 | - |
dc.title | Functional modules of DEMETER 5-methylcytosine DNA glycosylase necessary for seed development in Arabidopsis | - |
dc.title.alternative | 애기장대의 종자발달에 필수적인 DEMETER 메틸시토신 DNA 글리코실라제의 기능성 모듈 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | 임현정 | - |
dc.description.degree | Master | - |
dc.citation.pages | xi, 71 | - |
dc.contributor.affiliation | 농업생명과학대학 식물생산과학부(원예과학전공) | - |
dc.date.awarded | 2013-02 | - |
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