Analyzing The Openness Of Genome-Wide Chromatin Of Four Types Cells From Arabidopsis Using ATAC-seq

The male germ line in flowering plants divides asymmetrically to differentiate into haploid mononuclear microspores,producing a germ cell containing relatively small germ cells.Vegetative cells;germ cells undergo a second mitosis,producing two sperm cells.Vegetative cells and sperm cells control the growth of pollen tubes and the size of germ cells by activating different genetic and epigenetic mechanisms.The comprehensive characterization of this process relies on the effective separation of different types of cells throughout the development of male gamete development.Existing gene expression data based on whole genome sequences is growing,but little is known about how to establish,regulate transcription programs and define cell identity.Transcription factor(TF),the binding of specific DNA sequence motifs in the genome is an important mechanism of gene regulation.However,these sequences are hindered by the packaging of DNA into chromatin.The binding position and time of TF.Therefore,chromatin structure plays a key role in the regulation of gene expression.The role of acquiring and maintaining cell identity has received increasing attention.Since the combination of TF and cis-acting elements is usually associated with accessible chromatin regions.The ability to identify these accessible regions in the plant’s entire genome will improve understanding of the relationship between TF binding,chromatin status,and gene expression regulation.Transposase-accessible chromatin sequencing(ATAC-seq)is used to map open chromatin regions in animal genomes.In plants,the presence of plant cell walls,subcellular organelles,and the lack of stable transgenic lines limit the application of this technology.This article describes a method that combines ATAC-seq and fluorescent nuclear sorting to identify and locate the open chromatin regions and potential TF binding sites of four different types of Arabidopsis cell genomes.The difference in the degree of chromatin opening of different types of cells.The main findings are as follows:1.Obtain four different types of cells,including the nuclei of Arabidopsis mesophyll cells,pollen sperm and vegetative nuclei,mononuclear microspore nuclei,and male blasts.1)The nucleus of Arabidopsis mesophyll cells was extracted by the combination of enzymolysis and lysis,and the interference of mitochondria,chloroplast and other organelleswas removed.The results of DAPI staining showed that 90% of the nuclei were intact and of good purity;2)The crude extract of pollen grains was obtained from the trifle,and the pollen grains were crushed by the glass bead breaking method to release the sperm and vegetative nuclei.Two distinct cell populations were obtained by flow cytometry sorting,and further microscopic examination showed that high-purity sperm nuclei and vegetative nuclei were obtained;3)Microspores were roughly extracted from the flower buds of Arabidopsis thaliana and sorted by flow cytometry The microspores in the mononuclear period were obtained,and the microspore nuclei in the mononuclear period were further broken.The proportion of mononuclear microspores was 97.3%.The nuclei of mononuclear microspores were obtained by glass bead breaking.DAPI staining results showed that the purity and integrity of the nuclei were good;4)Separation from buds during meiosis In the pollen sac,male sex blasts are separated from the pollen sac,a certain amount of sex blasts are collected through the capillary tube,and a glass bead crushing method is used to obtain higher purity nuclei.The separation of the above-mentioned high-purity different types of cells has laid a solid foundation for further analysis of differential chromatin.2.The ATAC-Seq method was used to analyze the openness of chromatin in the genomes of different cell types and the differences in chromatin accessibility between different cells.First,construct a PCR amplification library to obtain fragments with a size between 200-500 bp.The detection of the microfluidic capillary electrophoresis system showed that the peak shape of the constructed ATAC-Seq library was wide,without obvious main peak,and the peak position was roughly 190-1000 bp.ATAC-Seq results show that the open chromatin regions of different cell types are concentrated near the transcriptional start site(TSS).Further inferring the chromatin accessibility region shows that more than 90% of the mesophyll nuclei are within 3 kb upstream of TSS,and less than 10% of the genes are located in distant intergenic regions.In addition,the accessible region of chromatin is also highly enriched in the TES region,which is consistent with some downstream regions required for transcription.According to the specific distribution of THSs,the specific distribution of the four samples is similar.They are mainly concentrated at 2K upstream of the gene,followed by 2K downstream of the gene and the intergenic region.These results indicate that most cis-regulatory elements are located in other types of Arabidopsis cells,and the regulatory region in the genome is mainly located near the core promoter of the gene.In summary,this study obtained high-purity cell lines of four different cells including Arabidopsis mesophyll cell nucleus,pollen sperm nucleus and vegetative nucleus,mononuclear stage microspore nucleus,and male sex cell Qualitative area identification.In order to further identify potential cis-acting elements and their associated transcription factors through differential open chromatin regions,especially the key differentially expressed transcription factors that are converted during reproduction,the dynamics of chromatin during male germ cell development are further elaborated Changes,laid the foundation for studying differential gene expression and transcription factor function.