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Interactional and Functional Characteristics on DNA Methyltransferase DNMT2 and Histone Deacetylase HD2 in Arabidopsis Thaliana

Author: SongYuan
Tutor: AnLiZhe
School: Lanzhou University
Course: Botany
Keywords: DNA methyltransferase DNMT2 The histone deacetylase HD2s family Protein interaction Gene Expression Arabidopsis Resilience mechanisms Epigenetics
CLC: Q943
Type: PhD thesis
Year: 2010
Downloads: 366
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Abstract


Epigenetics refers to the DNA sequence does not change, while the modification of a sequence change thereby affecting the phenotypic traits and genetic function of the organism. DNA methylation and histone deacetylation the classic epigenetic phenomenon, plays an important role in the regulation of the life process. The Plant Stress stress response regulation of epigenetic modification made great progress, research in this area is of great significance to the to clarify plant response molecular mechanisms Stress. In this thesis, biochemistry, molecular biology, genetics analysis and bio-informatics, transfer enzyme DNMT2 with histone deacetylase HD2s protein family interaction as well as physiological and biochemical functions of DNA methylation in Arabidopsis research the regulation of gene expression by microarray analysis, DNA methylation and histone deacetylation of cold stress analysis of the system and comprehensive exposition provides a rationale for the epigenetic regulation of plant stress tolerance The Lun basis. The results are as follows: 1. Colocalization Co-localization analysis bimolecular fluorescence complementation experiments Bimolecular fluorescence complementation analysis and in vitro co-immunoprecipitation pull down assay confirmed DNMT2 sub-cellular localization in the nucleus, in vivo and in vitro histone deacetylase HD2s family interaction. 2. GAL4/UAS system is used DNMT2 inhibition of gene expression by DNMT2 :: GUS fusion protein expression determination, of gene domain function in DNMT2, we identify and map the DNMT2 interaction and suppression area, gene inhibition zone near the C-terminus (aa177-384), can be effectively combined with targeted gene and inhibition of the expression. Near the N-terminus (aal-105) with HD2s interaction with histone deacetylase activity is closely linked. Analysis drought and cold stress, salt stress and the ABA on the Arabidopsis DNMT2, HD2s expression of cold stress HD2C expression is elevated The The expression of DNMT2 in 3 hours, there has been increased. Build pEarleyGate101-35S :: DNMT2, then transformed wild-type Arabidopsis (Col-0), get the overexpression DNMT2 transgenic plants, and the phenotype observed. Cultured for 20 days of the wild-type plants and 35S :: DNMT2 turn the genetically modified plants and 35S :: / / D2C (COE) of genetically modified plants and HD2C-mut (T99) is placed in the 0 ℃ different time periods, then ICE1 and COR1 gene expression The amount of analysis, found that certain HD2C cold-responsive genes. DNMT2 Although not directly involved in the activities, but may be important factors of indirect regulation of body activities. . HD2C different expression system: 35s :: HD2C transgenic overexpression plants (COE) and hd2c knock down mutant plants (T99) through class ELISA reaction and RT-PCR analysis of the DNMTs active, as well DNMT2 expression amount the expression of the same conclusion, showing a synergistic effect of the relationship. By immune coprecipitation Co-IP, and MALDI-ToF mass spectrometry analysis, DNMT2 potential interacting proteins were analyzed dehydrogenase guanylate cyclization DNMT2 may be related to the plant metabolic pathways. DNA methylation inhibitors (5-aza-2'-deoxycytosine) pretreatment plants, histone deacetylase inhibitors (trichostatin A) 0 ° C cold stress pretreatment plants and control plants, processing 24 hours later, the means of using gene chips, gene expression analysis, and diffraction in the database. Data analysis showed that DNA methylation and histone acetylation is not completely synergistic, even under cold stress, also generally present antagonistic relationship, only local collaboration, both apparent modification in the body homeostasis and complementary relationship. Based on the above results, we believe that DNA methyl transferase participation of enzymes DNMT2 in the of histone deacetylase HD2s gene family activities, with the function of inhibition of gene expression and regulation of plant growth and development. Gene chips for high-throughput analysis of gene expression, discussed the relationship of plant DNA methylation and histone deacetylation and antifreeze, and we hope to elucidate the epigenetic modification regulating mechanism, as well as for plant genetic engineering to improve crop tolerance to the ability to provide a certain basis, Lun.

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