Molecular Mechanisms Of Histone Demethylase Se15 Regulating Heading Date And Yield Components In Rice

The heading date of rice is mainly regulated by the internal and external environmental conditions such as photoperiod,air temperature and relative humidity,as well as the genetic factors within the crops themselves,which indicates that the plant is gradually transiting from vegetative growth to reproductive growth.Photoperiod is one of the most critical factors controlling rice heading and flowering,and two important factors causing rice flowering in photoperiod are Hdl and Ehd1.At present,the mechanism of Ehd1 pathway in rice is relatively clear,but the molecular mechanism involved in Hd1 pathway is relatively rare.In this paper,a mutant with early heading time was screened out from the gammaray mutagenicity of Gimbozu,and the phenotypic structure and genetic relationship of the mutant were analyzed,as well as the map cloning of Se15 gene.It was found that Se15 and JMJ706 were alleles participating in the regulation of epigenetic changes.We also found that Se15 and JMJ706 are alleles involved in epigenetic regulation.The results of the study are as follows.(1)Phenotypic observation:the Se15 mutant advanced the flowering period by 14 days compared with the wild type.(2)Genetic analysis and gene localization:Genetic analysis showed that the Se15 gene was controlled by a recessive nuclear gene.The gene was finely localized to a physical interval of about 1.2 Mb between MK10-13 and the end of chromosome 10 of rice.Generation sequencing of the gene within this interval revealed seven base deletion mutations in the coding region of the Os05g0577600 gene.(3)Complementation verification:expression of the Se15 gene was transferred to the mutant plants,and the mutant plants reverted to the wild-type phenotype at tassel stage.(4)Fluorescence quantitative PCR assay:The 24 h real-time expression of the main effector genes of the five tassels was examined under long-day and short-day conditions,respectively.The results showed that there was no significant difference in the 24 h real-time expression of the gene Ehd1 between the wild type and the se15 mutant under either long-or short-day conditions,indicating that Se15 may not be involved in the photoperiodic pathway of Ehd1 under both long-and short-day conditions,while the expression of the Hd1 gene in the se15 mutant under both long-and short-day conditions was significantly down-regulated from 8 to 24 h compared with the wild type.This suggests that Se15 may be an upstream gene of Hd1,which is involved in the Hd1-Hd3a pathway and thus regulates rice flowering period.(5)Validation of Se15 tasseling function in other varieties:Se15 knockout was completed in Nipponbare(NP)and Sasanishiki(Sasa)backgrounds,and the results showed that the mutant in Sasa background did not undergo tasseling alteration,while in NP background,tasseling was advanced by about one week.(6)Construction of se15-1,se15-2 mutants:The se15-1 with Jmj C structural domain was retained in the Sasa background,while the se15-2 mutant with deletion of ZnF and JmjC structural domains was found to have longer se15-1 grain length because of the deletion of ZnF,which in turn altered the increase in thousand grain weight.(7)Detection of histone methylation modifications:significantly increased modifications at the H3K9me2 site by hybridizing WT,se15-1,and se15-2 with H3,H3K27me2,H3K27me3,H3K4me2,H3K4me3,and H3K9me2.It was found that the modification of se15-2 at H3K9me2 site increased significantly.(8)RNA-seq analysis:seedling se15-1 expression was significantly down-regulated in OsABA8ox1 compared to WT,and se15-2 expression was significantly up-regulated in OsABA8ox1 compared to WT,this indicates that Se15 negatively regulates OsABA8ox1,ZnF and Jmj C structures,which may be involved in ABA pathway alone.In this study,we successfully cloned the tassel stage gene Se15 to reveal the molecular regulation mechanism of Se15 and found that ZnF of Se15 regulates rice grain size and participates in the ABA pathway at seedling stage,which provides an important germplasm resource for rice breeding applications.