Electrophoretic identification of restriction analysis products at the GATC site of wheat genomic DNA under salt stress

Abstract

Modern methods for the study of nucleic acids have shown the presence of modified nitrogenous bases in DNA, mainly, in the methylated form. Although many study were performed over the past few decades to elucidate the role of 5-methylcytosine, it has only recently been recognized that N(6)-methyladenine (6mA) - N6-methyladenine is present in quantitative and biologically active levels in the DNA of eukaryotic cells. It was established that 6mA content can vary by more than an order of magnitude within an isogenic population of organisms and 6mA levels in these organisms can be particularly sensitive to minor changes in the environment, for example, to stress factors. Methyl-specific restriction using Mal 1 endonuclease allowed to reveal changes in the adenylate methyl status of DNA in leaf cells of wheat under salt stress. This is a restriction enzyme that recognizes methylated adenine in the GATC nucleotide sequence and performs a symmetrical restriction at this site. The use of guanidine-isothiocyanate extraction allowed to isolate total DNA from leaf cells of wheat with virtually no signs of degradation, which is a necessary condition for further analytical studies. The use of the electrophoretic method, which ensures the separation of restriction fragments based on their charge and size, allowed to estimate the methyl state of the studied wheat leaf DNA sequence under different experimental conditions. Analysis of the results of restriction analysis of wheat DNA samples at different hours of salinity indicated that the distribution of restriction products based on their size significantly changes in the studied samples, which indicated a change in the methyl status of adenine in the GATC site. The results of densitometry indicate an increase in restriction products at the analysed sites. In comparison with the control variant, where mainly high molecular weight restriction fragments were found, at 24 h of incubation of plants in sodium chloride solution, the main share of products obtained by restriction with Mal 1 endonuclease was presented by low molecular weight fragments. An increase in wheat genomic DNA restriction products at the 24th hour of the experiment indicates a higher amount of methylated adenine in the GATC site during this period of the experiment. The changed in the distribution of adenine methylation may be a regulatory mechanism for the control of the adaptive response of cell metabolism in wheat leaves at the expression level of the corresponding genes.