| Kernel development is closely related to the yield,the mining of vital genes for kernel development and molecular mechanism analysis are the basis of high-yield molecular breeding in maize.In this study,the dek219(defective kernel219)mutant was derived from a natural mutation of maize inbred line W22,which displays opaque endosperm and embryo abortion.Through positional cloning and allelic test,we determine that Dek219 encodes a DCL1(DICER-LIKE1)protein,an essential enzyme in miRNA(micro RNA)biogenesis.Then miRNA sequencing,transcriptome sequencing and ATAC sequencing(assay for transposase-accessible chromatin using sequencing)were used to clarify that Dek219 affects chromatin accessibility by regulating miRNA synthesis,thereby regulating the expression of crucial genes in kernel development,which is a novel and vital regulatory pathway for maize kernel development.Gene expression analysis,protoplast transient expression analysis and EMSA were further performed to determine that the miR167h-Hsf17(Heat shock transcription factor17)-LSM10(Sm-like protein 10)module is one of the factors that affect the expression of histone genes and chromatin accessibility.Candidate gene association analysis showed that natural variations in Dek219,Hsf17 and LSM10 are significantly associated with kernel related traits and the expression of histone genes,and the related excellent natural alleles were mined.This study further improved the understanding of the regulation mechanism of maize kernel development,which is of great significance for improving maize yield.The main results were as follows:(1)The mutant exhibited stable phenotype of defective kernel,opaque endosperm,early embryonic lethality and no seedlings,in multiple environments.The kernel length,kernel width,kernel thickness and 100-kernel weight of the dek219 homozygous mutant were significantly reduced compared with the those of the WT(wild type).Paraffin section observation revealed that the early apical-basal axis of dek219 embryos were not established,and embryo development had been arrested before the early transition stage.Furthermore,the starchy endosperm cells of WT were more filled than those of dek219,and the ingrowths of WT were more dense.(2)The dek219 was mapped to the range to 120 kb physical region between markers Ind4.2 and Ind4.8 by positional cloning,this region contains six annotated genes.These six genes were amplified using PCR(polymerase chain reaction)and sequenced,sequencing analysis revealed that a T-base is inserted into exon 12 of Zm00001d027412,which causes a frameshift mutation,followed by a premature stop codon.The mutation resulted in the deletion of two essential RNase Ⅲ domains and two RNA-binding domains in Zm00001d027412.This gene encodes a DCL1 protein,a vital enzyme in miRNA biogenesis.(3)Candidate gene Zm00001d027412 was confirmed by allelic test,the mutation of the allelic material dek219-1 is a T-to-A substitution in exon 6,which results in the transformation of a tyrosine residue to a premature stop codon,resulting in the deletion of two RNase Ⅲ domains and three RNA-binding domains in Zm00001d027412.Ears from self-pollinated heterozygous dek219-1 exhibited segregated defective kernels at a ratio of25%,and the defective phenotype of these kernels were similar to that of dek219 kernels.Allelic test between heterozygous dek219/+ and dek219-1/+ generated ears exhibiting 3:1segregation.These results confirmed that Zm00001d027412 is the causative gene for dek219.(4)Loss of function of Dek219 resulted in significant decreases in the expression levels of miRNAs.A total of 59 differentially expressed miRNAs were identified between dek219 and WT.Among them,57 miRNAs were down-regulated in dek219 compared with WT,and only 2 miRNAs were up-regulated in dek219.After further screening,a total of 35 candidate miRNAs that may play a crucial role in maize kernel development were identified.(5)A total of 7,613 DEGs(differentially expressed genes)were identified between dek219 and WT,of which 2,836 genes were up-regulated and 4,777 genes were down-regulated in dek219 compared with those in WT.GO(Gene Ontology)term enrichment analysis suggested that the DEGs were most significantly enriched in nucleosome assembly,and the expression of most histone genes were significantly down-regulated in dek219.This may affect chromatin accessibility and genes expression,and may be the cause of defective kernel development in dek219.Furthermore,crucial genes for kernel development were also differentially expressed between dek219 and WT.(6)ATAC sequencing indicated that dek219 has higher chromatin accessibility than WT.By analyzing DEGs and differential ACRs(accessible chromatin regions)between dek219 and WT,we identified 119 candidate genes for maize kernel development that were regulated by chromatin accessibility.These include some reported vital genes for kernel development,these genes all have differential ACRs between dek219 and WT in their promoters.Differential ACRs in promoters can regulate the expression of these genes,and then affect maize kernel development,which is a novel regulatory pathway.(7)Through miRNA-target gene prediction and gene expression analysis,miR167h-Hsf17 module was considered as one of the candidate pathways for regulating histone gene expression.Hsf17 is a target gene of miR167 h,and miR167 h inhibits the expression of Hsf17 by transient expression analysis in protoplasts.Through phylogenetic analysis and gene expression analysis,we found that LSM10 gene may play an essential role in the regulation of histone gene expression and maize kernel development.Further protoplast transient expression analysis and EMSA verified that Hsf17 inhibited the expression of LSM10 by binding to TTCTAG motif.Further analysis confirmed that Hsf17 negatively regulated the expression of histone genes by inhibiting the expression of LSM10 gene.Our results indicated that the miR167h-Hsf17-LSM10 module regulates the expression of histone genes.(8)Candidate gene association analysis showed that natural variations in Dek219,Hsf17,LSM10 and histone genes were significantly associated with kernel related traits,which indicated that these genes were involved in the regulation of maize kernel development.Moreover,natural variations in Dek219,Hsf17 and LSM10 were significantly associated with the expression of histone genes,which indicated that these genes could regulate the expression of histone genes.In addition,excellent natural alleles and germplasm resources were identified,which provide important theoretical support and applied gene resources for the cultivation of improved varieties of high-yield maize. |
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