Big Data Analysis Of Transcriptional Regulation And Investigation Of Regulation Pattern Of DNA Methylation In Rice

Rice(Oryza sativa L.)is one of the main food crops and is the staple food for nearly half of the world’s population.Studying the biology related to rice is essential to improve the yield and protect the country’s food security.High-throughput RNAsequencing(RNA-seq)has become the most popular technology for profiling gene expression in the last decade due to its low cost and high coverage.As a result,the number of RNA-seq libraries from the plant community has been increasing exponentially in recent years.To take full advantage of the big-data of RNA-seq libraries,this paper constructs a comprehensive web-based platform,Plant Public RNA-seq Database(PPRD,http://ipf.sustech.edu.cn/pub/plantrna/).PPRD consists of 44,774 RNA-seq libraries,including rice(11,726),maize(19,664),soybean(4,085),wheat(5,816),and cotton(3,483).PPRD supports searches by gene ID,library ID,keywords,or any combination of these terms in selected libraries.PPRD will return multiple interactive diagrams,including expression levels among different tissues,developmental stages,abiotic and biotic stresses,and up-regulated or downregulated expression in mutant-related or treatment-related samples.PPRD also supports the query of gene co-expression,the built-in IGV browser to view the genomic mapping status,and the download of the expression matrix of genes in all libraries.The splicing of pre-mRNA is one of the essential steps in mRNA processing.Based on the Nanopore-based third-generation sequencing,this paper completed the sequencing of the full-length transcriptome of seedling in the elite hybrid Shanyou63 and its two parents,Zhenshan 97 and Minghui 63,including nuclear RNA and total RNA.Extensive post-transcriptional splicing was found in the Zhenshan 97 and Minghui 63.By comparing the post-transcriptional splicing levels of the identical alleles in the parents and hybrid,it was found that the post-transcriptional splicing levels of some genes were altered in the hybrid,and these differences may be due to the presence of different trans-acting factors in the hybrid.When comparing the posttranscriptional splicing levels between alleles in hybrids,it was found that the splicing levels of some alleles are consistent with those of the parents,while the splicing levels of other alleles in hybrids differed from the parents.These differences may be due to differential regulation of cis-acting elements in the hybrid.These results suggest that the regulation of intron splicing between parents and the hybrid is a complex process that requires the involvement of multiple factors for co-regulation.In addition,using a large number of rice RNA-seq libraries in PPRD,this paper found that different treatments specifically affect the post-transcriptional splicing process of different introns,and this dynamic regulatory process may help plants adapt the complex and variable environmental challenges.DNA methylation is one of the major forms of epigenetic modification of the genome,which plays a crucial role in regulating gene expression and maintaining the stability of the genome.Utilizing the highly efficient CRISPR-Cas9 genome-editing system,this paper created single-and multiple-knockout mutants for a lot of DNA methyltransferases in rice,including nine single-and eight multiple-knockout mutants.Whole-genome bisulfite sequencing revealed that OsCMT2,OsCMT3 a,and OsDRM2 are the three major players in the maintenance of non-CG methylation in rice.Os-dcc mutant shows a large number of differential expressions of TEs and genes.In addition,a high level of residual non-CG methylation exists in Os-dcc mutant,which features a high GC content.The residual methylation is lost in Os-ddccc mutant while retained in Os-ddcc mutant,suggesting that OsCMT3 b can maintain the nonCG methylation at GC-rich regions of Os-dcc mutant.Further analysis found that the highly GC-rich cluster of non-CG methylated sites seems to be unique for the rice genome,and OsCMT3 b is subfunctionalized to accommodate these specific sites.In summary,this paper constructs the PPRD,a plant public RNA-seq database containing about 45,000 public libraries,which supports rapid querying and downloading of gene expression in rice,maize,soybean,wheat,and cotton in all public RNA-seq data.Based on the Nanopore sequencing,this paper completed the full-length transcriptome sequencing of the elite hybrid rice Shanyou 63 and its parents Zhenshan 97 and Minghui 63,and systematically compared the cotranscriptional splicing rates and post-transcriptional splicing levels between two parents,between parents and the hybrid,and between alleles in the hybrid.In addition,this paper constructed a large number of rice DNA methyltransferases mutants by an efficient CRISPR-Cas9 genome-editing system,studied the function of methyltransferases and the relationship between different methyltransferases,and revealed the regulatory network of non-CG methylation in rice.