Identification And Characterization Of Histone H1 In Rice

Chromatin is a complex of DNA and protein found in eukaryotic cells.Nucleosome is the basic structural unit of chromatin,composed of histone and DNA.Histone H1 is a connecting protein connecting nucleosomes and DNA.As a basic protein of chromosomes,it is mainly involved in maintaining and stabilizing the high-level structure of chromosomes.Studies in mammals such as humans and mice have generally found that histone H1 has a significant inhibitory effect on the expression of most genes,and plays an important role in epigenetic regulation such as histone modifications and DNA methylation in heterochromatin regions.At the same time,H1 can cause gene silencing and also affect DNA methylation regulatory regions.At present,these research conclusions are mainly confirmed in mammals,and there is no related research and report on histone H1 in rice.In this study,we used rice cultivar Nipponbare as research material,cloned four histone H1 genes,constructed histone H1 fusion protein vectors driven by different promoters,and cultivated transgenic rice plants.A transient transformation system of rice protoplasts was established.Through RNA-seq,ChIP-seq and other omics-related technologies,histone H1 in rice and its relationship with gene expression were systematically analyzed.Related research results help to better understand the regulatory function of rice histone H1 at the genome wide level.The main findings are as follows:1.Four genes annotated as histone H1 were found in the rice gene database and publications,namely LOC_Os04g18090(OsHl.l),LOC_Os06g04020(OsH1.2),LOC_Os03g58470(OsHL3),LOC_Os07g08710(OsH1.4).And sequence alignment was performed with the sequences of histone H1 in human and Arabidopsis thaliana,it showed that the rice histone H1 is closely related to the Arabidopsis histone H1 and far from the human histone H1.2.Total RNA were extracted from Nipponbare,and the coding sequences of H1 were cloned.The expression vectors of H1 fusion protein,which were driven by CaMV35S promoter were constructed.Infect the rice callus by Agrobacterium-mediated method respectively,after co-cultivation,hygromycin resistance screening,pre-differentiation,differentiation,rooting culture and other processes,more than 20 independent transformants were obtained respectively Identification showed that about 70%of them were positive transgenic plants.Transgenic T1 plants were obtained by self-crossing,and it was found that the grain type of the T1 generation changed significantly,the grains became significantly slender,and the 1000-grain weight decreased.3.The expression vectors of H1 fusion protein,which were driven by rice Ubi promoter were constructed.Establish a rice protoplast transient expression system,using rice protoplasts as materials for subcellular localization and Western Blot experiments.The results show that histone H1 is localized in the nucleus and H1 protein levels can be successfully detected in protoplasts.4.OsH1.1 was transfected with protoplasts and analyzed by high-throughput transcriptome sequencing,4 samples(the control group and the overexpression group were each sequenced twice).7.5 Gb clean data was obtained after sequencing quality control.Clean data were aligned to the rice reference genome and expression level of each gene was calculated,the expression levels of the control group and overexpression group of rice OsH1.1 were 134.178 and 51638 respectively.We screened 3721 differentially expressed genes,of which 1669 genes were significantly up-regulated and 2052 genes were significantly down-regulated.Combining gene function annotation,GO and KEGG analysis,it was found that changes in OsH1.1 expression mainly affected photosynthesis and pyrophosphatase activity.OsH1.1 overexpression also affects the expression of some grain shape-related genes.