Screening And Functional Analysis Of DNA Methylation Regulating Factors Response To Abiotic Stress

In eukaryotes,DNA methylation is an important epigenetic marker in the regulation of gene expression.In plants,DNA methylation occurs in all three cytosine contexts,namely CG,CHG,and CHH(H represents A,T,or C).DNA methylation can be inherited to the offspring,regulating the gene expression,cell differentiation,gene imprinting and embryonic development of the organism.A large number of studies show that,DNA methylation keeps dynamic change by establishing,maintaining and removing DNA methylation.The proteins that regulate DNA methylation in plants are mainly DNA methyltransferases which establish DNA methylation,DNA demethylases which remove DNA methylation,and some proteins which can help them take functions.In addition,the modification of DNA methylation is also influenced by histone modifications and chromosome structure.However,little is known about the upstream regulatory factors of DNA methylation and demethylation.In the natural environment,there are many abiotic stresses.Abiotic stress seriously affects the growth and developmment of crops,and it is a major factor in the decline of crop production in the world.Although the signal transduction pathway of abiotic stress has been studied,whether DNA methylation and demethylation modification are involved in this regulation is still not very clear.The mechanism of DNA methylation response to abiotic stresses is also need to be elucidated.Through a series of experiments,we have proved that SAG(Sensitive to ABA during Germination)is involved in the regulation of DNA methylation level.The results of the study are as follows:(1)Screen DNA methylation regulatory factors in response to stress by CHOP-PCR.CHOP-PCR was used to screen the Arabidopsis mutants which response to stress.It was found that the DNA methylation of SAG T-DNA insertion mutant(sag)in DT-77(AT1G26400 Promoter Region)was significantly increased.(2)The DNA hypermethylation of sag mutants was confirmed by bisulfite sequencing.(3)Promoter sequence analysis revealed that AtSAG promoter region contained LTR and other stress and hormone cis acting elements.SAG gene encodes a functional protein of AtPDS protein family.The full-length cDNA corresponding to the SAG mRNA is 2622 bp in length and encodes a putative protein of 873 amino acids.We predict that SAG contains LBR-Tudor domain(Amino Acids 602–658).The LBR-Tudor domain of SAG are highly conserved in Arabidopsis and in other species.(4)Complementary lines using SAG promoter can complement the phenotype of sag.(5)Through the analyses of DNA methylation regulator genes expression,we found that the transcription level of DNA demethylase gene ROS1 is significantly down regulated,which consists with the genomic DNA hypermethylation levels.(6)SAG is localized in the endoplasmic reticulum.Arabidopsis transformed with an expression plasmid for the SAG–GFP fusion protein exhibited GFP fluorescence in the endoplasmic reticulum.(7)SAG protein can interact with ADP1.In order to understand how SAG functions,we analyzed the date of immunoprecipitation(IP)followed by liquid chromatography-tandem mass spectrometry(LC-MS/MS).We found that 132 proteins may interact with SAG.Yeast two hybrid experiments showed that SAG protein can interact with ADP1.