Studies On The Function Of EFD In RdDM Pathway In Arabidopsis

DNA methylation is a conserved epigenetic marker in plants,mammals,and some fungi,mainly refers to the transfer of methyl groups on S-adenosine methionine to DNA cytosine under the catalysis of methyltransferase,which plays an important role in the epigenetic processes,such as transcriptional silencing of transgenic and endogenous genes,secondary mutation,imprinting and X chromosome inactivation.In plants,there are about one-third of the methylated DNA sites associated with small RNAs—called RNA dependent DNA methylation(Rd DM),and Rd DM is considered to be a ubiquitous transcriptional silencing mechanism in plants.This process consists of several proteins that process and produce si RNAs,modify histones,modify chromatin and methylate cytosine in all sequences.These proteins mainly include DNA methyltransferases,histone modification enzymes,chromatin remodeling proteins and plant-specific RNA polymerases.Although DNA methylation contributes to stable,long-term and heritable silencing,in some cases the level of DNA methylation can be rapidly altered by the mechanism of active DNA demethylation,a rapid transformation of DNA methylation and demethylation that more dynamically regulates genomic stability.In plants,active DNA demethylation is primarily performed by a small group of bifunctional DNA glycosylase,including ROS1,DME,DML2 and DML3.EFD(Exine Formation Defect)is a gene found in our laboratory that plays an important role in the early regulation of pollen,three-dimensional structure analysis of the protein revealed that EFD has a typical structure of DNA methyltransferase,and enzyme activity experiments in vitro also verified its DNA methyltransferase activity.The discovery and study of EFD is of great significance because few methyltransferases are known in plants related to reproductive development.This paper aims to explore whether EFD play the catalytic role of methyltransferase through Rd DM mechanism,and whether there is an interaction between EFD methyltransferase and ROS1 demethyltransferase family.The results obtained so far are as follows:1.In order to study the role of EFD in the Rd DM process,we constructed the Target and Silencer vectors containing α promoter,which were respectively transferred into arabidopsis,and the homozygotic and stable dual vector system was screened by fluorescence and PCR,known as the Target/Silencer system.2.we cross efd with Target/ slislient system,and then select the homozygous.In addition,gene mutants(drd1,dms3,drm2)of the known Rd DM elements were selected and hybridized with the Target/ slislient system to screen out homozygous lines as the positive control group.Sulfite sequencing results showed that EFD had no direct role in the Rd DM process of α promoter,but an indirect role.For further verification,the fluorescence of mature cotyledon embryos was observed,and the results were consistent with the methylation level.3.To further validate the role of EFD in the Rd DM process,we got seeds containing the RD29A-LUC system.Through hybridization,we obtained RD29A-LUC homozygous lines with and without mutation of EFD,Sulfite sequencing results showed that EFD had no obvious direct or indirect effect on Rd DM of RD29 A promoter.4.Relationship between EFD and ROS1 and its homologous gene DML2、DML3.We purchased ROS1 and its homologous gene mutants,after the mutants identification,we detected the methylation levels of at1g26400,ERT1,ERT2 and Z3 in the corresponding mutants,respectively.The results showed that EFD did not regulate the ROS1 target gene at1g26400,ROS1 and DML3 cooperated with EFD to regulate the methylation levels of ERT1,ERT2 and Z3 sites.Another homologous protein DML2don’t seem to regulate ERT1 and Z3 sites,but it could play a synergistic role at the ERT2 site.The subsequent real-time fluorescence quantification and Hpa II enzyme digestion results were basically consistent with the methylation detection results.