Studies On The DNA Methylation Function Of EFD And Its Homologous Genes In Arabidopsis

Epigenetic regulation is the change in gene function without alteration of DNA sequence,and this change is heritable.There are several regulation modes: DNA methylation,nucleosome localization,chromatin remodeling and histone modification.DNA methylation refers to the transfer of methyl groups on S-adenosylmethionine to DNA molecule under the catalization of DNA methyltransferase.In general,this change occurs on cytosine.DNA methylation needs to be maintained by DNA methyltransferase.When DNA methyltransferase lacked,the promoter of some genes could be highly demethylated,which leads to the high expression of the genes.It means that DNA methylation is of great significance to organisms.DNA methylation occurs in CG,CHG,CHH(H = A,T or C)sites in plants.DNA methyltransferases in mammals are divided into two types: de novo methyltransferases and maintenance methyltransferases.There are three main types of DNA methyltransferases in plants: maintenance methyltransferase MET1,which is mainly responsible for maintaining CG methylation;de novo methyltransferase DRM2,which is mainly responsible for de novo methylation;and plant-specific methyltransferase CMT3,which is mainly responsible for maintaining CHG methylation.EFD is a gene related to early anther development in Arabidopsis thaliana found in our previous research.The corresponding efd mutant had the phenotype of early anther abortion.Bioinformatic analysis revealed that EFD had the similar three-dimensional structure with typical methyltransferases,suggesting that EFD encodes a DNA methyltransferase.Homology comparison showed that there are five EFD homologous genes in Arabidopsis which also have methyltransferase domain,indicating that they may also have methyltransferase activity.The purpose of this study is to study the expression pattern of EFD homologous genes and the phenotypes of the single mutant,to find out target sequences of EFD and homologous genes,to study their methyltransferase activity,and to compare EFD and homologous proteins with typical DNA methyltransferases to explore their similarities and differences.The results we obtained are as following:1.The expression pattern analysis of EFD homologous genes showed that the homologous genes were expressed in Arabidopsis anthers,roots and leaves.In vitro pollen germination experiments showed that the pollen germination rate of the single mutant of EFD and homologous gene was lower than that of the wild type,but there was no obvious abortion of silique.2.Bisulfite sequencing analysis indicated that under the background of EFD and homologous gene single mutant,the methylation levels of ERT1 and ERT2 commonly detected in the literature of DNA methyltransferase were significantly lower than that of wild type.q RT-PCR analysis found that ERT1 and ERT2 in the mutants were significantly up-regulated,suggesting that ERT1 and ERT2 were target sequences of EFD and homologous genes.The methylation-sensitive endonucleases-PCR assay proved that ERT3 and ERT4 were also target sequences of EFD and homologous genes.3.Previous results of in vitro enzyme activity test showed that EFD may have the function of de novo methyltransferases.In order to detect the biochemical function of EFD homologous proteins,we constructed the expression vector of EFD homologous gene and expressed of EFD homologous protein in vitro.After methylation sensitive endonuclease assay and bisulfite sequencing experiments,we found that EFD homologous proteins probably do not have the function of de novo methyltransferase of DNA.4.To study the methylation function of EFD and homologous genes in vivo,We obtained the ert1 mutant and crossed it with the single mutant of EFD or its homologous genes.The methylation level and the expression level of ERT1 were detected in F2 plants which has the same genotype as wild type.The results showed that the methylation level of ERT1 did not increase,indicating that the methylation level of ERT1 was not restored by EFD or its homologous genes to that of wild type.At the same time,we constructed the EFD gene complementation vector and transferred it into the efd mutant.The methylation level of ERT1 in the complementation plants did not increase,which also indicated that EFD did not restore the methylation level of ERT1.5.Previous experimental results showed that EFD and its homologous genes do affect the methylation of some genes.In order to compare the similarities and the differences of them with the typical methyltransferases in plants,we detected methylation levels of MEA-ISR?ATSN1?LTR2?ERT1?ERT2?ERT3 and IBM1 genes.The results showed that all genes were not regulated by EFD or its homologous genes except ERT1,ERT2 and ERT3.