Effect Of 5-azaC On The Development And DNA Methylation Of Wheat(Triticum Aestivum L.)

Plant DNA methylation plays essential roles in plant genome and development because it is correlated to epigenetic variation and has important function in genome defense,gene expression regulation and so on.DNA methylation inhibitor 5-azacytidine(5-azaC)has been used in the studies of plant epigenetics that based on DNA demethylation.Some usefully epigenetic traits that were related to undermethylation were taking potential breeding value.The germination status,seedling morphogenesis and growth indexes in the field after 5-azaC treatment of wheat cultivars were studied,meanwhile genetic stability and DNA methylation variation of the samples were initially researched by AFLP and MSAP.The main results are as follows:1.To study the effect of methylation inhibitor 5-azaC on the germination speed and regularity of wheat,wheat seeds were treated with different concentrations of 5-azaC respectively.The results showed that the germination ability of wheat was promoted by 5-50μM methylation inhibitor,and the germination speed and regularity of wheat treated with 50μM 5-azaC were better than controls and other treatments.However,the germination ability could be inhibited by high concentration of 5-azaC in some degree.2.The development of the samples treated with 5-azaC and the controls were observed.No seedling died when germinating seeds were treated for 10 days.The development of roots and seedlings treated with light concentration 5-azaC were similar to controls'.However,50μM azaC had affected root's morphogenesis.Nevertheless the growth of wheat's roots and seedlings were all significantly inhibited by 5-azaC over 100μM.The inhibitory action has dose adding effect. Meanwhile,after treated with different concentrations 5-azaC,the growth indexes of wheat seedlings had different changes when growth in the field.The tiller ability increased to some extent after treatment with different concentrations 5-azaC(except 0.5μM).Some concentrations not only promoted heading and flowering but also affected other growth indexes such as plant height,1000-grain weight.While the effect was relate to the concentration of 5-azaC.3.The genome DNA of treatments and controls were analysed by AFLP technique.The DNA was isolated from the leaves of 10-day-old samples treated by 5-azaC and controls.A suitable concentration of DNA was digested with restriction enzyme(EcoRI and MseI),and 2930 clear bands were scored using 7 pairs of primers in 9 samples.No polymorphic bands were found among different samples treated with 5-azaC.The results revealed that no nucleotide sequence had altered during 5-azaC treatment.4.The DNA methylation status of samples was analysed by MSAP.Ten pairs of primers amplified about 460 clear bands,compaired to the control,some methylation alterations were found in the samples after 5-azaC treatment.The DNA demethylation bands was over 60%of the total variation fragment.It is clear that the main role of 5-azaC is to make DNA methylation decrease. The level of DNA demethylation was increasing when 5-azaC concentration was adding. Demethylation levels were increase with 5-azaC concentration,the maximum methylation variation bands that was up to 47 were seen in 1000μM 5-azaC treatment's leaves and the variation rate was as high as 10.0%.Meanwhile,the patterns of methylation vatiation was consistent in different treatments.