Construction And Function Analysis Of Gmddm1 Mutant Using CRISPR/Cas9 System In Soybean

Epigenetics refers to heritable changes in gene expression or phenotypes without altering the nucleotide sequence of the gene.DNA methylation,as an important regulatory mechanism of epigenetics,is critical for gene regulation and genomic stability.DDM1(DECREASED DNA METHYLATION 1),is an ATP-dependent SWI2/SNF2 chromatin remodeler that is essential for maintaining whole-genome DNA methylation levels.In Arabidopsis,the methylation of the ddm1 mutant was reduced by 70%without apparent morphological phenotype.In maize,rice and tomato,dysfunction of DDM1 leads to hypomethylome with a strong deleterious phenotype.However,how DDM1 interacts with other methylation pathways and the specific function of DDM1 protein is unclear.Therefore,in this study,we constructed a Gmddml mutant using CRISPR/Cas9 technology,performed methylation analysis and phenotypic detection on the soybean mutant,in order to provide a reference for the molecular mechanism research of DDM1 function in plants,concluded as follows:1.Bio informatics shows that the DDM1 gene is quite conserved,with two highly similar copies in the soybean genome,with the DEXDc domain and the HELICc domain.The similarity between GmDDM1a and GmDDM1b gene was 94.882%,where the length of CDS region of GmDDM1a gene was 2286bp encoding 762 amino acids,and the length of CDS region of GmDDM1b gene was 2289bp encoding 763 amino acids.DDM1 is highly conserved in the plant kingdom.The phylogenetic tree analysis shows that the GmDDM1 protein has the closest relationship with the DDM1 protein of the leguminous family,and can be clearly divided into monocotyledonous and dicotyledonous.2.Soybean Williams 82(W82),Jack and Dongnong 50 were used as experimental materials.RT-PCR showed that DDM1 gene played an important role in soybean,which had certain tissue specificity and expression differences among different germplasm resources.The regulation of DDM1 expression is related to the state of the cells.The higher the expression of the more active cells,the more relevant it is to genetic differences.3.The knockout vectors were successfully constructed by CRISPR/Cas9 technology.Twenty-three heterozygous mutants and 50 homozygous mutants were identified by genetic transformation.All six homozygous mutants are base deletion mutants,resulting in frameshift mutations and pre-stop condon whole of translation.4.After methylome analysis we found that the soybean mutation Gmddml affected the DNA methylation level of the genome.The methylation level of Gmddml in CG and CHG sites was decreased.Gmddml a-1C,Gmddm1a-2C and Gmddm1b-2C have increased methylation levels at the CHH site,and Gmddmlb-2C is particularly evident.The DMRs features analysis showed that DMRs were mainly distributed in the intergenic region of the CHH locus.Compared to wild,Gmddml mutant show a dwarf morphorlogies with more branches,leading to decreased yield per plant.