The Effect Of DNA Demethyiation On Tolerance Of Arabidopsis Thaliana To Low Temperature Stress And The Construction Of Differential-methylation Subtractive Library Of Arabidopsis Thaliana In Response To Low Temperature Stress

DNA methylation is an important mechanism of epigenetic regulation and plays a key role in plant development and gene expression regulation.DNA methylation/demethylation occurs when plants are exposed to environment stresses.In the present study,we used Arabidopsis to study the role of DNA demethylation in plant response to low temperature stress.In order to identify genes regulated by methylation mechanisms under cold stress,we developed a protocol which combines methylation-sensitive restriction endonuclease(MS-RE)digestion with suppression subtractive hybridization(SSH)to construct a differential-methylation subtractive library.The main results were summarized as follows:1.DNA demethylation treatment resulted in increased tolerance to low temperature in Arabidopsis seedlings:1)DNA demethylation treatment increased the survival rate and decreased the LT50 of Arabidopsis after freezing treatment.2)DNA demethylation treatment decreased the MDA accumulation in Arabidopsis cells,suggesting that DNA demethylation treatment can protect membrane from low temperature damage.3)By increasing the activity of antioxidation enzymes,DNA demethylation treatment decreased cell damages induced by low temperature stress.4)DNA demethylation treatment caused increased content of small molecular compounds such as soluble sugars and proline in cells,which make plants more tolerant to low termperature.2.NO production may involve in the increase of tolerance to low temperature resulted from DNA demethylation treatment:1)DNA demethylation treatment increased the NO content in Arabidopsis cells.2)Application of exogenous SNP(NO donor)didn't affect the increase of tolerance to low temperature resulted from DNA demethylation treatment.However,application of PTIO(NO scavenger)reversed the protection of DNA demethylation-treated plants from low temperature damage.3)Transcription analysis showed that DNA demethylation treatment promoted the expression of NIA1 and P5CS1.3.In this study,we constructed a differential-methylation subtractive library of Arabidopsis in response to low temperature stress by combining methylation-sensitive restriction endonuclease(MS-RE)digestion with suppression subtractive hybridization(SSH)for identifying genes regulated by methylation mechanism under cold stress.We further verified the efficiency and reliability of the liabrary.1)The overall effiency of the library was 79.27%.2)By sequencing some clones of the library,75 gene fragments were obtained.The functions and distributions in genome of these 75 genes were analyzed.3)DNA methylation analysis demonstrated that the methylation status in some of above-mentioned genes was altered after low temperature stress.4)Transcription analysis indicated that DNA demethylation induced by low temperature or 5-Aza treatment promoted the gene expression level.