Genetic Study On The Effect Of Rice Histone OsH1.3 On Agronomic Traits In Rice And Its Application

In eukaryotes linear DNA molecules are folded into chromatin and stored in the nucleus,and nucleosomes are the basic structural units that make up chromatin.Although histone H1 does not form nucleosomes,it is a key protein in maintaining nucleosome structure,binding to DNA on the outside of core histones and anchoring it at the entry and exit points of nucleosomal DNA.Thus,histone H1 maintains the stability of nucleosomes,prevents chromatin remodelling from occurring and is important for the formation of higher chromatin structures.Current studies in Arabidopsis thaliana have found that histone H1 affects plant growth and development,suppresses gene expression and plays an important role in epigenetic modifications.These findings have also been validated in mammals,but few reports have been made on histone H1-related functions in rice.In this study,we performed subcellular localization of histone H1 in a rice protoplast transient expression system;constructed a transgenic vector and grew stable transgenic plant monocots;examined the expression of histone H1 at the RNA and protein levels of transgenic plants;identified the phenotype of transgenic plants;analyzed the changes of histone modifications in transgenic plants,and analyzed the regulatory mechanism of histone H1 in rice by phenotypic analysis,transcriptome analysis and histone The main results of this paper are as follows.The main findings of this paper are as follows:According to previously published literatures and rice public database search,four rice histone H1 proteins were identified:LOC_Os04g 18090,LOC_Os06g04020,LOC_Os03g58470,and LOC_Os07g08710.Homology analysis was conducted among four rice histone H1 and three Arabidopsis histone H1.The phylogenetic tree showed that the rice and Arabidopsis H1 shared relatively high homology at the protein level.Among them,LOC-Os04g 18090 falls into the same evolutionary clade with Arabidopsis histone ATH1.3,so it was named as OsH1.3.A subcellular localization vector of CaMV35S-OsH1.3-GFP fusion protein was constructed.The subcellular localization of histone OsH1.3 was studied in rice protoplast transient expression system and observed using confocal microscopy after transformation.The result confirmed that OsH1.3 was accumulated in the nucleus.The OsH1.3 gene fused with the flag tag was overexpressed under the control of the OsH1.3 endogenous promoter,and OsH1.3 transgenic lines were created.Transgenicpositive plants were verified by PCR with the flag tag primer,and 4 lines were selected for further study,named as OsH1.3-1,OsH1.3-2,OsH1.3-3,and OsH1.3-4.Subsequently,the expression changes of OsH1.3 in transgenic plants were detected,and it was found that the endogenous OsH1.3 expression level was significantly increased in OsH1.3-1,OsH1.3-2,and OsH1.3-3,while the endogenous OsH1.3 transcription level in OsH1.3-4 was similar to that of Nipponbare.The total protein extracted from OsH1.3 transgenic plants was then subjected to Western Blot using Anti-Flag antibody to determine whether the OsH1.3-Flag protein was expressed.Observing the phenotype of OsH1.3 transgenic lines during rice growth in the field,it was found that the OsH1.3 transgenic plants had obvious phenotypic differences compared with Nipponbare.In the OsH1.3 transgenic plants the plant height became shorter,the tillering number decreased,the pollen fertility decreased,the seed setting rate decreased,the seed length and grain width were shortened.RNA-seq analysis was conducted by using the anthers of OsH1.3 transgenic plants,and transcriptome sequencing was compared to Nipponbare.It was found that the expression levels of genes LOC_Os12g21850,LOC_Os10g40290,LOC_Osl2g37350,and LOC_Osllg25560,which are related to pollen fertility,were altered in the OsH1.3 materials.Additionally,ATAC-seq experiments revealed changes in open chromatin in OsH1.3 transgenic plants.Western blot analysis was performed to detect changes in histone modification in OsH1.3 transgenic plants,and it was found that the enrichment level of the repressive modification H3K27me3 was increased.Therefore,ChIP-seq experiments were therefore performed with leaves of OsH1.3 transgenic plants and the nuclei of the leaves were extracted,and the results showed that H3K27me3 modification were selectively enriched at specific genomic sites in OsH1.3 transgenic plants,resulting in an overall increase in the enrichment of H3K27me3 and affecting the phenotype of the plant.