Construction And Application Of Target DNA Demethylation System Based On CRISPR/dCas9 In Plants

DNA methylation plays an important role in plant growth,development and stress response.Additionally,it is also a potential source of genomic variation.In plants,DNA methylation based flowering time and the other complex traits regulations have been identified through population genetics researches.Howerver,there is no effective means to precisely operate the DNA methylation status of sequence specific sites,which greatly limits the further functional research and utilization of these sites.In our study,CRISPR-dCas9 protein was fused with ROS1 protein with DNA demethylation function to form a DNA demethylation system targeting specific sequences.In order to improve the efficiency of DNA demethylation,we constructed the fusion protein of phage capsid protein dimer(MS2)and ros1,and designed a g RNA site with specific hairpin structure that can bind to MS2.Target DNA methylation specific sites in Arabidopsis and maize protoplasts,The main results are as follows:1.In order to test the efficiency of DNA demethylation editing based on dCas9-ros1 system,the promoter hypermethylation region of FWA gene in Arabidopsis was selected as the target to express the vector in Arabidopsis protoplasts.Through the bisulfite BS sequencing of the transformed target sequence,it was found that the DNA methylation level of the target region was significantly reduced,which proved that the dCas9-ros1 system had the function of target DNA demethylation.2.DNA hypermethylation on the promoter of SKP1 INTERACTING PARTNER3(SKIP3,AT2G02350)gene was detected in Arabidopsis epiril population in the early reference period.Therefore,CRISPR / dCas9-ros1 system was stably expressed in Arabidopsis plants by Agrobacterium mediated inflorescence staining.In transgenic positive plants,DNA methylation of target site was significantly reduced,while the expression of SKIP3 was significantly increased.Four pairs of other genes not related to flowering were randomly selected as controls.It was found that the expression of these genes in transgenic plants was not significantly different from that in wild-type Arabidopsis plants.It was proved that dCas9-ros1 DNA methylation editing system could effectively edit target sites and had a certain specificity.Through the phenotype identification of Arabidopsis transgenic positive plants,it was found that with the decrease of DNA methylation level of SKIP3 promoter,the flowering period of plants was significantly delayed,and the number of leaves increased.In order to further clarify how SKIP3 affects plant flowering.We analyzed the expression of FLC,CO,FRI,CCA1 in transgenic plants and found that the expression of FLC in transgenic plants increased significantly.3.In the early stage of our research group,we screened out the heterotopia of DNA methylation difference of different drought tolerant inbred lines by sequencing the genomic DNA methylation of drought tolerant inbred line AC7643 and drought sensitive inbred line AC7729/TZSRW.After detecting the variation of DNA methylation level of candidate sites Chr8:176652465-176652814 in AC7643 and AC7729/TZSRW under normal water condition and drought stress,it was found that under PEG simulated drought stress,the DNA methylation level of target region in maize roots decreased significantly.The DNA methylation level of drought sensitive inbred line AC7729/TZSRW was significantly higher than that of drought tolerant inbred line AC7643.This site was selected as a g RNA target to construct the dCas9-ros1 vector and transform it in the protoplast of maize inbred line AC7729/TZSRW.Transient expression of dCas9-ros1 resulted in a significant reduction of DNA methylation level in the target region and a significant increase in the expression of the corresponding gene Zm00001d012560.The results further showed that dCas9-ros1 system could modify DNA methylation level of specific target sites in dicotyledons and monocotyledons represented by Arabidopsis and maize.4.In the related population constructed by 200 maize inbred lines,using general linear model,mixed linear model and Bayesian inference respectively,the population DNA methylation level was regarded as genotype,and the whole genome association analysis was conducted with the plant survival rate after seedling drought stress,and42 DNA methylation sites controlling the tolerance of Maize seedling plants to drought stress were screened together.Among them,the DNA methylation level of MAP sequence(epi MAP)located in 32144650-32144770 bp region of chr1 chromosome was significantly related to the plant survival rate under drought stress in different models.The higher the DNA methylation level,the lower the plant survival rate and the fewer the number of root branches of the corresponding inbred line under drought stress.In RIL population,under drought stress,the transcription level of root target MAP gene decreased significantly.Meanwhile,it was detected that the expression level of natural antisense transcripts MAP corresponding transcripts downstream of the gene increased significantly.The DNA methylation level in the first exon of natural antisense transcripts decreased significantly after drought stress.Based on Fickett algorithm,the protein coding potential of map corresponding transcripts was analyzed.Fickett score was 0.45,significantly lower than the average score of coding protein(0.74);According to the previous RIL population parent ribosome profiling,there was no distribution of reads in this transcriptional region,so it was speculated that the map corresponding transcripts were lncRNA.However,the relationship between DNA methylation and gene expression is not clear.Specific g RNA was designed based on epi MAP site sequence and expressed in maize protoplasts instantaneously.It was found that the decrease of DNA methylation level promoted the expression of lncRNA.