Study Of The Epigenetic Regulatory Mechanisms On Temperature And Red Light Responsive Gene XCT In Arabidopsis

Gene expression is mainly influenced by the chromatin structure, which is governed by processes often associated with epigenetic regulation, including non-coding RNA, chromatin remodeling, histone modification and DNA methylation. DNA methylation is an important epigenetic mark for the regulation of gene expression in eukaryotes. RNA-directed DNA methylation （RdDM） is an important regulatory phenomenon involved in repressive epigenetic modifications that can trigger transcriptional gene silencing （TGS） under abiotic stress in plants. The gain or loss of DNA methylation is correlated with a considerable decrease or increase in the corresponding amount of mRNA abundance and with the presence or absence of24-nt siRNAs at each silenced epiallele. Increasing evidence has shown that the activity of siRNAs could be triggered by various environmental stimuli to affect the targeting chromatin structure.Cold stress and heat stress are the main limiting factors for affecting crop yields and distribution. Plant has already formed the mechanism of resisting the cold and heat stress. Although classical genetic approaches in cold and heat stress have been studied deeply, whether DNA methylation,24-nt siRNA and histone modifications participate in the regulation process is still not clear.This research use the bioinformatics and experimental techniques of molecular biology to identify possible important functional gene of response the heat and cold stress related epigenetic regu\ation（AT2G21150, AT3G49240, AT4G13660, AT4G21580）, and found that the promoter regions exist DNA methylation regulation.（1） By analyzing the database of GENEVESTIGATOR, promoter analysis of the selected genes was then carried out in Arabidopsis epigenome maps and siRNA data base website to identify hypermethylated regions.17genes（AT2G20610, AT4G18440, AT4G36500, AT4G13660, AT1G25988, AT4G21580, AT1G02270, AT4G22740, AT1G21050, AT5G02320, AT2G07696, AT3G10940, AT3G49240, AT3G45300, AT5G45930, AT2G21150, AT2G39800） may affected by cold and heat stress and epigeneitic modifications were selected for further study.（2） By using quantitative real-time reverse transcription-PCR （qRT-PCR） and the DNA methylation sequencing （BSP）, we found the promoters of four candidate genes （AT2G21150, AT3G49240, AT4G13660, AT4G21580） had the changement of methylation modification and the transcription levels were induced by stress.（3） The two T-DNAmutant lines（SALK₁08639C、SALK₁34513） of gene AT2G21150showed the hypersensitivity to heat stress at germination stage, the germination rate and percentage decreased obviously. These results revealed that AT2G21150may participate in heat stress resistance pathway during seed germination.（4） By analyzing the XCT gene promoter region, a lot of methylation sites and histone modifications were dected.（5） By using the q-PCR and BSP sequencing methods to verify XCT redlight induced the DNA methylation changes as well in the gene promoter region, the conclusion is that the red light does induce XCT gene expression and Correlated with change of the DNA methyl-ation.（6） By siRNA targeting prediction, XCT gene promoter region could be targeted2924-nt siRNAs, q-PCR experiments showed that the accumulation of siRNA significantly reduced after the red light treatment. It proved that RdDM regulation pathway was involved in the response of XCT to red light.