DNA Methylation Difference Between Day And Night In Populus Nigra Detected By MeDIP

The circadian clock is an endogenous timekeeper that allows organisms to anticipate the day/night cycle in nature environment.From parts of prokaryotes to all the eukaryotes,this endogenous mechanism enhance their growth and fitness by coordinating numerous biological events with the environment.In plants,the clock has been shown to regulate a wide variety of processes,including hypocotyl and root growth,flowering time,sugar metabolism,photosynthesis,nutrient homeostasis,hormonal signaling,and immunity.Researches have shown that circadian clocks in higher plants is a complex regulatory network,affecting a number of biochemical and molecular signaling pathways.In this study,the global methylation patterns in mature leaves of one-year-old Populus nigra N46 at 8:00 and 24:00 were detected by MeDIP-seq(Methylated DNA immunoprecipitation sequencing),different DNA methylation profiles were obtained and the possiblity of DNA methylation in plant circadian clock regulation were discussed.The main results are as follows:1.different genomic DNA methylation profiles between day and night in poplar was discovered in this study.The global methylation level in mature leaves of P.nigra N46 were detected by MeDIP-seq method,and a totaol of 11.16 G clean data was obtained in 8:00 and24:00,with mapping rate of 48.12% and 49.28% respectively.Sequences with methyl-cytosine were distributed in different genomic regions,and the promoter regions had the ratively higher peak frequencies than other genomic regions.Results showed that there were more down-methylated peaks in sample of 8:00 than that of 24:00;Most of the up-mehtylated peaks distributed in promoter regions,while down-methylated peaks often distrubuted in other genomic regions.Therefore,differen methylation patterns were found in genome of P.nigra N46 between day and night.2.a total of 160 differential methylated genes were obtained between samples of 8:00 and24:00 and biological function of those genes were analysed by GO and KEGG.The GOenrichment results showed that most of the differential methylated genes participated in the constitude of cells and organelle parts,and their molecular functions were related to binding and catalytic activities,and involve in biological processes including cellular process,metabolic process,response to stimulus,death and localization.The KEGG pathway analysis indicated that differential methylated genes were mapped to carbohydrate metabolism process,nucleotide metabolism process,genetic Information processing,environmental adaptation and signal transduction pathways.3.the methylation level of several differential methylated genes were validated and mRNA expression level were detected by RT-qPCR.BS-PCR results showed that the methylation level of five differential methylated genes were consistent with the MeDIP-seq results,indicating that the MeDIP-seq results were reliable.The m RNA expression level of six differential methylated genes with methylation changes in promotors and exons were detected by RT-qPCR.Results showed that the the expression of CHSL2 gene,Histidine phosphotransfer protein gene and Multicopper oxidase gene,that had high methylation level in24:00 samples were deceased compared with that of in 8:00 samples;whereas the expressin level of PTRMTP2 gene,GluR5 gene and Nbs-lrr resistance protein gene that had low methylation level in 24:00 samples were increased.These results indicated that the methylation changes in promoter and coding region did influnce the expression level of those genes,similar to results of previous studies.