Effect Of5-azaC On DNA Methylation And Gene-expression Of Chrysanthemum

In this study, the chrysanthemum outdoor seedlings of “Quan xiang chong tian” were treated with0μM,100μM,200μM,400μM5-azaC solution in vivo processing respectively. In the development ofchrysanthemum, the blade shape and plant height and flowering-time were observed and recorded.Physiological index of Chlorophyll and malondialdehyde content were determined. In addition to, we usedmethylation sensitive amplified polymorphism (MSAP) and high performance liquid chromatography(HPLC) test DNA methylation level and mode. And the relationship between phenotypic change and thegenomic DNA methylation of chrysanthemum was studied Used cDNA amplified fragment lengthpolymorphism (cDNA-AFLP) to analysis genome-wide expression. The aim was to tried to reveal themechanism of DNA methylation which effect on the growth and gene expression of chrysanthemum. Theexperimental results as follows:(1) Phenotype: The leaves of treatment groups were curled after treated for30d compared to control,and there was positively correlated between curled degree and5-azaC concentrations. we found that5-azaCchrysanthemum plants dwarf after treated for30d. Four groups of materials were significant differencebetween plant height, and had a dose of the cumulative effect. After untreated for20d, plant height of100μM recovered approach to control, but200and400μM groups restore slower, still has the remarkabledifference to control. Flowering time was related to the concentration of5-azaC, but the relevance not theregularity of positive correlation or negative correlation. The flowering-time of four treatment groups weredifferent. The flowering-time of100μM and200μM advanced9and16days respectively compared tocontrol. There was no difference of observation pattern among the three groups when the flowers fully open.These suggested that ornamental value was not affected although the chrysanthemum was treated with5-azaC. The flowering-time of400μM treatment group delayed about3days. This suggested that a highconcentration of5-azaC has inhibitory effect.(2) The detection of physiological indexes: there are closely relationship between phenotypic variationand physiological reaction, so the relevant physiological indexes were tested. Chlorophyll content of eachsample: chlorophyll content of100μM and200μM were slightly higher than the control group, however,400μM were lower than control. The Malondialdehyde content of400μM was higher than other three groups. Low concentration5-azaC had little Effect on physiological reaction of chrysanthemum, but a highconcentration5-azaC had certain harm to the cell.(3) DNA Methylation detection: genome DNA of chrysanthemum leaf which by the concentration of100,200,400μM methylation inhibitors of5-azaC processed and unprocessed as material, using MSAP andHPLC analysis of genomic DNA methylation changes. The results showed that he methylation level ofthree treatment groups were lower than the control group, and the reduce magnitude increased with theincrease of concentration of5-azaC, and the trend was leveling off.(4) Genes-expression analysis: the concentration of100,200,100μM of methylation inhibitors5-azaCprocessed and unprocessed chrysanthemum leaf total RNA of material. The results as follows: compared tothe control group, methylation proportion of100μM reduced by12.91%;200μM reduced by19.16%;400μM reduced by23.75%. This shows that the level of DNA methylation was lower and lower with theconcentration of5-azaC increased. And the reducing DNA methylation pattern in some site of the otherthree groups was consistent, about accounted for4.80%. Using cDNA-AFLP to analyse potted seedlings,and24primer combinations amplified772TDFs, there was different TDFs between each treatment groupand control group the treatment group, of which100μM treatment group has25up-regulation expressionTDFs;200μM treatment group has30up-regution expression TDFs;400μM treatment group has22up-regution expression TDFs.10TDFs were sequenced successfully of16TDFs which between100-400bpwere cloned, and7of which displayed homology to genes with known functions,3no had a match.Functional analysis indicated that7TDFs mainly participated in3kinds of processes of signal transduction,protein degradation and synthesis, stress responses.