Using CRISPR/Cas9 Gene Editing Technology To Construct Gmros1 Mutant And Analysis Of Gene Function In Soybean

Epigenetics refers to the heritable changes in gene expression levels based on non-gene sequence changes,including DNA methylation,histone modification,and non-coding RNA.A specific DNA methylation state is an outcome of dynamic regulation by de novo methylation,maintenance of methylation and active demethylation,which are catalysed by various enzymes that are targeted by distinct regulatory pathways.Active demethylation triggered by DNA demethylase.REPRESSOR OF SILENCING1(ROS1)with glycosylation and lyase activities plays an important role in plants growth,development,biotic stress and abiotic stress response.In this study,we constructed a gmrosl mutant using CRISPR/Cas9 gene editing technology,after methylation detection and phenotype observation,which made a foundation for exploring the biological function of ROS1 in soybean.The main results are as follows:1.Three GmROSl copies were found in soybean based on gene homology.Glyma.03G190800,Glyma.10G065900 and Glyma.13G151000 were named GmROS1L,GmROS1A and GmROS1B,respectively,based on gene structure and evolution analysis.After multi-alignment,it was found that GmROS1L,GmROS1A,and GmROS1B are highly conserved with the ENDO3c domain and the PFAM domain.The phylogenetic tree analysis shows that GmROS1L had the closest genetic relationship with a copy of MtrROS1L in alfalfa;GmROS1A had the closest genetic relationship with GmROSIB.2.Soybean Dongnong50(DN50)was used as experimental materials.RT-PCR showed that the expression of ROS1 gene was tissue-specific in soybean.The highest expression level of GmROS1L were in flowers,similar to the expression pattern of AtROSl;The highest expression level of GmROS1A and GmROS1B were in seeds,which is similar to the expression pattern of DME in the DNA demethylase family.3.The gene knockout vectors of GmROS1L?GmROS1A and GmROS1B were successfully constructed by CRISPR/Cas9 gene editing technology respectively.After PCR,sequencing and dCAPS verification,one homozygous mutant of GmROS1L were identified through cotyledonary node pathway-mediated genetic transformation.named gmros11-12C.There are lbp cytosine missing in position 729 of the exon which create a mutation from isoleucinea to stop codon in the 247th amino acid.4.After methylome analysis we found that the soybean mutation gmrosll affected the DNA methylation level mainly on the transposon region.The methylation level of gmrosll in CHH site was increased.The differential methylation regions(DMRs)of gmros11-12C were mainly distributed on CHH locus.And it is found that the number of hypoDMRs(30443)is significantly more than hyperDMRs(14839).5.Compared with the wild type,mutants showed more branches,smaller internodal length,smaller leaves,and increased 100-seed weight.The plant height and number of main stem nodes did not change significantly.