DNA Methylation Of Transgenic Male Sterile Mutants In Arabidopsis Thaliana

Male sterility is widespread in higher plants,and it is one of the most important approaches to crop heterosis utilization.Male sterility is associated not only with the lack of viable pollen,but also with the failure of pollen release.Arabidopsis thaliana has a large number of male sterile mutants,which can provide a good experimental material for the detailed study of flower development,microspore production,pollen differentiation,anther dehiscent and its gene function.Brassica napus and Arabidopsis thaliana are both Brassicaaceae plants,therefor the mechanism of male sterility in Arabidopsis thaliana can provide highly reference value and the theoretical foundation for the utilization of Brassica napus heterosis.In this study,genomic DNA methylation sequencing and small RNA sequencing were performed on Arabidopsis thaliana transgenic anthers indehiscent mutants M6 and M24.These data were analyzed by bioinformatics,and the relationship between the mutant anthers and the methylation and miRNAs was explained.The main findings are as follows:1.The genome methylation level of the wild type,mutant M6 and M24 was 11.5%,12.7% and 12.4%,respectively.The whole genome methylation level of the mutant showed an increasing trend compared with the wild type.To further analyze the methylation level of the gene coding region and the promoter region,we found that the mutations in the CHH sequence of the mutant were much more than the other two sequences,indicating that mutations at M6 and M24 could be related to the RdDM pathway.The results of the experiment were consistent with:the mutations rdr2-2,dcl3,drm1/2,nrpd1a-3,nrpd1b-11 on the RdDM pathway as the parent and M6 and M24,respectively,and the F1 phenotype is restored.2.This study compared the M6 and M24 differential methylation genes and found 269 hyper-CHHme differential methylation genes.Among them,11 genes were found to be associated with flower development,such as SAUR-like,F-box family-related genes.RT-PCR was used to verify the candidate genes.It was found that the CHH methylation in the gene body was increased and the expression level was decreased,which was consistent with the data analysis.3.We performed small RNA sequencing of M6 and M24 mutants and analyzed with DNA methylation data.Compared with the expression of miRNAs in wild type and mutant,we found that miRNAs with different M6 and wild type were 9,and there were 11 miRNAs with different M24 and wild type,and ath-miR398b-3p,ath-miR398c-3p,ath-miR8175,ath-miR408-3p,ath-miR869.2 are miRNAs common to both mutants.We found that elevated levels of CHH methylation accompanied by 24 nt-sRNA elevation were associated with genes associated with flower development,F-box / RNI-like superfamily protein,SADHU3-1.This conclusion further suggests that mutants M6 and M24 are small RNA-mediated DNA methylation enhancement,leading to a decline in the expression of some of the flower-related genes,resulting in anthers indehiscent.