Analysis Of Promoter Methylation Map From Calli And Transcriptome From Stem Cell In Arabidopsis Thaliana

In vitro organogenesis refers to the process that the plant tissue or cell mass differentiates to form roots,shoots or plantlets due to the totipotency of plant cells.Recent studies reveal that DNA methylation plays important roles in plant in vitro organogenesis.However,the functions of DNApromoter methylation in plant regeneration are largely unknown.Plant stem cells are undifferentiated cells which can not only renew themselves,but also provide precursor cells for the formation of tissues and organs.Until now.most studies focus on the animal stem cells.In the in vitro culture system,the mechanism of maintenance and differentiation of plant stem cells are still unclear.In this study,we compared the DNApromoter methylation maps of NC and S-NRC.Combined with the results of their transcriptome,the candidate genes were selected,which might function in shoot regeneration under the regulation of promoter methylation.Detailed analysis revealed that there was a negative correlation of DNApromoter methylation level and the expression level of candidate gene PRF which played important roles in shoot regeneration.Moreover,we established the transcriptomes of stem cells and non-stem cells by using the Arabidopsis stem cell marker line pCLV3::GFP::GUS,and screened the candidate genes that were expressed specifically in the stem cells.The above results would lay the foundations for the studied of stem cell maintenance and differentiation.The main contents and results are as follows:1.Functions of DNA promoter methylation in shoot regeneration1.1 Obtaining of embryonic and non-embryonic calliIn the medium containing 2,4-D,the seeds of Col-0 produced calli which obtained the characteristic of embryo.And on the other,we used the non-embryonic calli of our lab to obtain protoplasts,and finally got the non-embryonic calli originated from single cell.1.2 The establishment and analysis of promoter methylation patterns of RC and NRC1.2.1 Promoter methylation comparison of RC and NRCBy comparison of the promoter methylation of RC and NRC,we found that obvious difference existed between two types of materials.About 34%genes were different in the methylation level.1.2.2 Gene expression profile analysis between RC and NRCBy GO analysis,we found that differentially methylated genes mainly participated in the biological process development regulation,transcription regulation,auxin and cytokinin response,biological metabolism,transportation,lipid metabolism,protein synthesis,transportation and modification,and stress response and so on.In total,there were 6395 differentially methylated genes and 238 were probably involved in shoot regeneration,which participated in hormone response,cell differentiation and proliferation,and plant development.1.2.3 Conjoint analysis of promoter methylation and expression levelsBy conjoint analysis of promoter methylation and expression levels,we found 234 genes whose expression levels were negatively related to methylation level.We then selected 24 genes which might be related to the regulation of shoot regeneration.1.2.4 Candidate genePRF affected shoot regenerationWe used the demethylation mutants of PRF to identify' its shoot regenerationability compared to the wild type.We found that its ability was reduced and at the same time its expression level was up-regulated.Then we predicted that the high expression of PRF was responsible for the reduction of shoot regeneration.1.2.5 Expression patterns of PRF during the subculturing of calliWith the purpose to identify the correlation of the expression of PRF and the maitainence of embryogenic calli,we used the mature seed of the Arabidopsis,and screened poor-grown,unwound calli.After screening and subculturing for generations,the differentiation ability of the calli reduced gradually.By analysis of the expression of the PRF,we found that the expression level rised with the differentiation ability reduction of the calli.Then we predicted the lose of embryogenic character were related to the high expression of SRF.2.Stem cell sorting and transcriptome analysis2.1 Obtaining of calli of pCLV3::GFP::GUS?pWUS::GFP::GUS andpSTM::GFP::GUSUsing the suspension culture system,we induced the embryoid of pCLV3::GFP::GUS?pWUS::GFP::GUS and pSTM::GFP::GUS.Then we used GUS activity to identify the expression of WUS,CLV3 and STM.We found that CLV3 had the highest expression level while WUS and STM had very low expression level.So we selected pCLV3::GFP::GUS as the metarial to sort protoplasts of stem cells.2.2 Protoplasts of pCLV3::GFP::GUSBy FACS(Fluorescence Activated Cell Sorting),we sorted and collected the cells with GFP(Green Fluorescence Protein)and the cells without GFP.Then we used these cells for transcriptome sequencing.2.3 Analysis of transcriptome between GFP cells and non-GFP cellsBy analysis of transcriptome between GFP cells and non-GFP cells,we found that 359 genes were up-regulated and 1328 were down-regulated in GFP cells.GO analysis revealed that these genes participated in the biological process development regulation,transcription regulation,auxin and cytokinin response,biological metabolism,transportation,lipid metabolism,protein synthesis,transportation and modification,and stress response and so on.We selected 44 genes for further studies,including 27 hormone response genes and 17 meristem-related genes.