| Rice false smut,caused by Ustilaginoidea virens(teleomorph Villosiclava virens),is an important disease in rice planting areas worldwide.U.virens infects rice spikelets and converts grains into smut balls whose color changes from gray to yellowish and ultimately to greenish black,that is also their chlamydospores color changes on sporodochia of the ball.That is,the transforms non-dormant yellowish chlamydospores into dormant greenish black ones.This phenomenon is epigenetic.The phenotype of these asexual spores in color and dormancy is obviously different.In order to study this difference which is related to DNA methylation,profile of DNA methylation levels and pattern and the difference fragment of three forms of non-dormant conidia,yellow chlamydospores and dormant black chlamydospores in U.virens were performed by a methylation sensitive amplification polymorphism(MSAP)method and sequence analysis of specific fragment.The main results are as follows.(1)In order to establish the experimental material adapted to DNA methylation,the mixture of conidia suspension and mashed mycelium from single spores of U.virens was used for the analysis.The yellow and black chlamydospores were isolated at different development stages of U.virens,and the conidia were isolatedfrom the single spore strain which cultured on Threat of the philosophy's media,150 r/min,28?for 7 days.There were 5 methods,i.e.CTAB\SDS and the kit A\B\C,being applied in this study to extractgenomic DNA of the conidium,the yellow and black chlamydospores of U.virens.Relatively,the kit C method is the best method for extracting high quality genomic DNA.(2)MSAP analysis was performed to analyze DNA methylation level difference among non-dormant conidia,yellow chlamydospores and dormant black chlamydospores in U.virens.The results showed that total methylation rate of conidia genomic DNA CCGG sequence was 20.56%,the yellow chlamydosporeswas 33.52%,and the black chlamydospores was 25.63%.These results indicate the DNA methylation level among them exists difference,and DNA methylation level of dormant black chlamydospores is lower than non-dormant yellow chlamydospores.Based on detection of the amplified DNA bands of the conidia,yellow and black chlamydospores,the fully-methylation rate of these 3 samples was 8.73%,17.68% and 13.96%,that is,the fully-methylation rate of yellow chlamydospores > black chlamydospores > conidia.While their hemi-methylation rate was 11.83%,15.84% and 11.67%,respectively.That is,the hemi-methylation rate of yellow chlamydospores > conidia > black chlamydospores.(3)Seventy one variant MSAP fragments were identyied from conidia,yellow chlamydospores and dormant black chlamydospores in U.virens.Then all of these fragments were sequenced.Based on Blastn and Blastx analysis in NCBI website,we found that the conidia virant fragment are homologous to Hydrolytic enzyme,DNA binding regulator,alpha amylase,and so on.On the other hand,the virant fragments of yellow chlamydospores(non-dormant)are homologous to DNA-binding transcriptional regulator and ATP-dependent protease.They regulate the complicated growth and differentiation processes during cell development,which keeps cells playing normal physiological functions.In addition,the variant fragments of the black chlamydospores(dormant)are homologous to peptidyl-prolyl isomerases,histidine binding enzyme,methyl coenzyme M reductase alpha subunit,ABC-type polar amino acid transport system,and so on.These virant sites are methylated and inhibit gene expression.For example,maybe peptidyl prolyl isomerase can inhibit cell reproduction,which results in cell dormant.Through this study,the difference of the DNA methylation level detected among non-dormant conidia,yellow chlamydospores and dormant black chlamydospores of U.virens.Based on the reported functions of variant framgments' homologes,the DNA methylation differential genes in conidia,yellow chlamydospores and dormant black chlamydospores probably regulate the spore dormancy in U.virens. |
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