| Chrysanthemum?Chrysanthemum×morifolium Ramat?is one of the most important ornamentalplants in the world.It has many economic values,such as ornamental,edible,medicinal and tea.Chrysanthemum originated in China with 1600 years history.The diversified variation is seldom seen among other cultivated plants.Therefore,the origin of this variation has become one of the most impotant problems aroused general concern nowadays.DNA methylation is one of the most well-studied epigenetic modifications and involves many biological processes,including transposon silencing and activation,gene imprinting and chromosome inactivation,which plays an important role in the regulation of plant growth and development.Whether DNA methylation plays a role in the formation of various mutant traits of chrysanthemum needs to be discussed.The identification and analysis of the DNA methylation modification enzyme genes of chrysanthemum is an important basis for this problem.In the study,using the chrysanthemum cultivars?Jinbeidahong?,transgenic chrysanthemum materials?Zijingling?and Chrysanthemum×nankingense?C.nankingense?as the experimental materials.Studying on DNA methyltransferase and demethylase genes analysis of using bioinformatics methods,real-time fluorescence quantitative PCR and other methods.In addition,transcriptome sequencing of MET1-RNAiNAi material revealed the effect of DNA methylation reduction on chrysanthemum growth and development at the transcriptional level.The main results obtained are as follows:?1?The full-length transcript data of‘Jinbeidahong'was obtained by library sequencing usingRNA from chrysanthemum mixed tissues?roots,stems,leaves,petioles,foot buds,lateral buds,flower buds,petals,stamens and receptacles?,and the‘C.nankingense'genome data was downloaded from Chrysanthemum Genome Database?http://www.amwayabrc.com?.The DNA methyltransferase and demethylase genes of‘Jinbeidahong'and‘C.nankingense'were initially identified by conserved domain and phylogenetic tree analysis.In the‘Jinbeidahong',10 DNA methyltransferase and 5 demethylase gene transcripts were identified;in the‘C.nankingense',10 DNA methyltransferase and 5 demethylase gene were identified.The expression of chrysanthemum in different tissues and varieties were analyzed by real-time fluorescence quantitative PCR.Compared with root,CmMET1,CmCMT2 and CmDRM2 genes have differential expression in leaf were significantly higher at the seedling stage.At the flowering stage,CmMET1,CmCMT2,CmDRM2 and CmDME genes have similar expression patterns.Compared with root,the expression levels from high to low were the stem,root,flower and leaf,and in stem were extremely significant.The expression levels of CmMET1,CmCMT2,CmDRM2 and CmDME genes were different among 10 different chrysanthemum varieties.The expression patterns of DNA methyltransferase and demethylase genes in the root,stem and leaf of‘C.nankingense'at seedling stage were quite different,which indicated that different tissues have different methylation patterns.In conclusion,the identification and analysis of DNA methyltransferase and demethylase genes will contribute to the study of the role of DNA methylation in the growth and development of chrysanthemum.?2?The MET1-RNAiNAi obtained in the previous group was used as the material,and the stem andleaves of the seedling stage were taken.Three biological replicates were processed and controlled in each part,and the transcriptome was sequenced using the Illumina HiSeq 4000 sequencing platform.The results showed that:1)A total of 82.28Gb Clean Data was obtained from 12 samples.The Clean Data of each sample reached 6.26 Gb,and the percentage of Q30 base in each sample was not less than 90.12%.2)After assembly,a total of 100,800 unigenes were obtained.The 43,476 unigenes were obtained through functional annotation,among which 23,179 unigenes were over 1kb in length.3)The 8 differentially expressed genes?DEGS??8 up-and 0 down-regulation?were found in MET1-RNAiNAi leaf compared with the control group leaf,which were three Viral RNA helicase Viral,two RNA dependent RNA polymerase,one carlavirus coat and one 0.7kD viral coat protein.There were 156 DEGS?145 and 11?in the MET1-RNAiNAi stem and the control group stem,encoding many key proteins in plant biology processes,such as transcription factors,signal transduction mechanisms,biosynthesis,transport and catabolism of secondary metabolites and interactions of plant pathogens.In the control group leaf and stem,8786 DEGS?3461 and5325?were involved in carbohydrate transport/metabolism,post-translational modification protein turnover/chaperones,secondary metabolites biosynthesis,transport and catabolism,transporter activity,catalytic activity and so on.The 8323 DEGS?3195 and 5128?in the MET1-RNAiNAi leaf and MET1-RNAi stem,which involved in carbohydrate transport/metabolism,post-translational modification protein trunover/chaperone,signal transduction mechanism,amino acid biosynthesis,catalytic activity,transcription factors and so on.4)A real-time fluorescence quantitative PCR was used to verify the expression levels of seven differential genes,and the results were basically consistent with the sequencing results. |
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