| DNA methylation is one of the most important epigenetic modification which is stable inheritance and conservatism in plants and animals.And it also has tissue specificity and developmental stage specificity.Studies have found that genomic DNA methylation of plant cells often changes after the differentiation of different tissues.However,the study about the changes of genomic DNA methylation and its effects on gene expression is very limited in the process of differentiation and dedifferentiation.What's more,the pattern and distribution of genomic DNA methylation in different plant has species specificity.There is a lack of attention to whether species specificity has an effect on gene expression during cell differentiation or dedifferentiatioa In this study,we took typical oil plant castor bean as experimental materials,performed genomic DNA methylation and transcriptome high-throughput sequencing on embryo and embryo callus,to analyse DNA methylation change law in the process of tissue culture of castor bean and its effects on gene expressioa These results would provide some insights on DNA methylation diversity of different plant tissues,but also have important significance for understanding the biological function of DNA-methylation.The main results are as follows:1.In order to obtain the callus of embryos,we first optimized the tissue culture system of embryo callus,the embryos of the radicle and cotyledon are excised on the medium for inducing callus,and observed the status of callus induction by different hormone ratios by setting TDZ and NAA with different concentration gradients.It was found that when the concentration of 6-BA and NAA were 2 mg/L and 0.3 mg/L,respectively,and the pH was 5.8,the callus status of embryos induced on MS medium was the best,and the incision begins to swell a few days later.After one month,callus is produced successively,and the rate of healing is over 90%.The callus grows well and presents hard,light yellow particles or small pieces that can be used as subsequent analysis.2.To investigate the variation of DNA methylation in embryos and callus,we performed whole-genome DNA methylation sequencing.We found that CHH methylation:isdominant,accounting for more than 50%,while the percentage of CG and CHG methylation was small.On the whole genome,the level of CG methylation was significantly higher in the body region than in the upstream and downstream 2 kb regions;as for CHG and CHH methylation,their methylation levels in gene body were lower than the upstream and downstream 2 kb regions.Differential methylation analysis of embryo and embryo callus resulted in 35287 differentially methylated regions(DMRs),including 1349 CG DMR,608 CHG DMR,and 33330 CHH DMR.In CHH DMR,99.8%of DMR washypomethylation in embryo and callus.In general,this suggested a downward trend during callus culture.3.In order to investigate the difference in gene expression between embryo and callus,we performed transcriptome sequencing and obtained a total of 18274 genes.Of them14,563 genes were expressed both in embryo and embryo callus,1998 genes were only expressed in embryo and 1713 genes were only expressed in callus.A total of 11,704 genes were differentially expressed,and 90.7%(10626)were up-regulated in callus,and only a small fraction(1078)were down-regulated.Among these differentially expressed genes,we identified 19 genes that may be involved in callus formation,including six hormone-related differentially expressed genes,six genes involved in the cell cycle,and three genes involved in wound induction,and four genes involved in reacquisition of embryonic fete.4.To investigate whether DNA methylation has an effect on gene ex:pression regulation,we performed a correlation analysis between differentially expressed genes and DMR.The results showed that the expression levels of4363 genes were negatively correlated with their DMR methylation level At the same time,among these related genes,we identified four key genes involved in callus formation,and the expression level was significantly negatively correlated with DNA methylation.When methylation occurs in the body region ofthe gene,the degree of DNA methylation is significantly negatively correlated with the corresponding gene expressio.When DMRs occur in the promoter region and the 2kb region downstream of the gene,DNA methylation and gene expression are also present Negative correlations,these results reflect a significant negative correlation between hypomethylationof embryonic callus genomic DNA and gene expression.In conclusion,this study investigated the variation of DNA methylation and the differences in gene expression at the transcrptome level between castor embryos and post-differentiation embryo callus.Based on the association analysis of DNA methylation and differentially expressed genes,it was found that DNA methylation was all reduced in the embryogenic callus,and the expression of related genes was significantly increased,showing a significant negative correlational the genome level. |
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