DNA Methylation Research Of Oryza Sativa(Nipponbare) After Treated With 5-Aza-2’-deoxycytidine

DNA methylation plays a key role in embryonic development, genetic transcription, X chromosome inactivation and cell carcinogenesis. The DNA methtylation patterns and levels are regulated by the function of DNA methyltransferase and DNA demethylase. The DNA demethylation includes active DNA demethylation and passive DNA demethylation, which are controlled by the DNA demethylase and methyltransferase, respectively. So far, lots of work are focus on the DNA methyltransferases, it will be very important and useful to study on the DNA demethylase genes regulating of gene spacial and temporal expression. In this thesis, the DNA demethylase gene copies were identified from the genomes of O. sativa, S. bicolor, A. thaliana and P. trichocarpa. Then the homology, structure, expression and evolutionary relationships of them were analysed, and the relationship between their evolution and function were explored. On the other hand, although the differences of the function of DNA methyltransferase genes have been clarified clearly through the studys of mutants, to what extend of the tissue culture and DNA methylation inhibitors affect on the DNA methylation and phenotype are still inconclusive. In this thesis, rice seeds were treated and the callus were induced with different concentrations of DNA methylation inhibitors (5-Aza-2’-deoxycytidine,5-Aza-dC), and then to detect the DNA methylation level of specific sites and explore the DNA demethylation role of 5-Aza-dC.1.We identified 8,6,7,6 DNA demethylase genes from the genomes of O. sativa, S. bicolor, A.thaliana and P. trichocarpa respectively based on 10 known DNA demethylase genes. The copy number of genes is very similar, which are random distributed, while most of them are colineariry on the chromosomes of different species. The DNA demethylase genes have a conservative glycosidase domain that contain 149 amino acid. The DNA demethylase gene family can be divided into four subfamilies (ROS1, DML3, DML4 and DML5) based on the conservative glycosidase domain with maximum likelihood method, and DML4 and DML5 are newly identified.2. Although the numbers of a-helic and β-pleated sheet of secondary structure of each DNA demethylase are different in rice and Arabidopsis, the secondary structure of DNA demethylase are very similar, suggested that some demethylases could have similar function. The expression of DNA demethylase genes are tissue specific.It has lower expression in rice endosperm than in other organs, Therefore, we speculated that major DNA methylation in endosperm is passive demethylation, not active demethylation.3.5-Aza-dC can inhibit the growth of rice callus, and has lethal effect under high concentration. Moreover, the regeneration plants from the callus appear dwarf phenomenon, while plants phenotypes have no obvious change when seeds dealing with 5-Aza-dC. The results suggested that the demethylation function of 5-Aza-dC could be different under different treatments.4. Tissue culture can decrease the DNA methylation level, which was observed in the promotor of RPBF gene and reached similar DNA methylation level to the endosperm, while it doesn’t affect on the DNA methylation of RISBZ1 gene. It has the DNA demethylation function on RISBZ1 gene only at the certain concentration of 5-Aza-dC (10.0 μM), while it doesn’t affect on RPBF gene. Interestingly, it could promote the DNA methylation of RPBF gene in the callus induced under the certain 5-Aza-dC concentration (2.5μM), but not RISBZ1 gene, suggested that tissue culture and 5-Aza-dC has locus-specific demethylation. Meanwhile,5-Aza-dC might have the function of DNA methylation, which needs to be verified by experiment.5.5-Aza-dC and tissue culture can activate the retransposition of retransposon Tosl7 by DNA demethylation, but it has no effect on the DNA methylation of Tos17Chr10,suggested that the reactive retransposon is the copy of Tos17Chr7 not Tos17Chr10.