Rice Genomics and Genetics 2024, Vol.15, No.1, 28-35 http://cropscipublisher.com/index.php/rgg 31 Figure 4 presented the process of identifying the genome-wide direct targets of the OsMYB8 protein and its functional analysis. a) The nuclear localization of OsMYB8-GFP in rice protoplasts was visualized by co-localization with D53-mCherry nuclear marker. b) Yeast two-hybrid experiments showed that full-length and truncated OsMYB8 proteins fused with the DNA-binding domain (BD) exhibited transcriptional activation activity. c) The core binding motif "TTHGGY" of OsMYB8 protein was identified using MEME-ChIP tool. d) Venn diagram compared the genes bound by DAP-seq technique with differentially expressed genes (DEGs) identified by RNA-seq in Osmyb8 mutants. e) GO (Gene Ontology) enrichment analysis of 345 overlapping genes, with Molecular Function (MF) and Biological Process (BP) represented in different colors indicating levels of P-value, and the size of circles representing the number of genes in each functional category. f) ChIP-qPCR analysis showed that OsMYB8 could bind in vivo to the promoter region of OsJAR1, with the red line indicating the position of the "TTHGGY" motif in the OsJAR1 promoter region. g) Electrophoretic mobility shift assay (EMSA) demonstrated that GST-OsMYB8 recombinant protein could directly bind to the OsJAR1 promoter region containing the "TTHGGY" motif. h) Transient dual-luciferase (LUC) reporter gene assay indicated that OsMYB8 could induce the transcription of OsJAR1 promoter in rice protoplasts, with the LUC/REN ratio representing the relative activity of the OsJAR1 promoter. Figure 4 Identification of the genome-wide direct targets of OsMYB8 Therefore, the results further confirmed the key role of OsMYB8 in regulating rice diurnal floret opening time (DFOT), especially through its direct action on downstream target genes like OsJAR1, thereby affecting the JA-mediated signaling pathway and related biological processes. Moreover, these findings also revealed the important role of transcription factors in plant growth and development and response to environmental changes. Figure 5 detailed the role of the OsJAR1 gene in regulating the diurnal floret opening time (DFOT) in rice and its relationship with the content of jasmonoyl-L-isoleucine (JA-Ile). a) Using CRISPR/Cas9 gene editing technology, Osjar1 mutants were created in the ZH11 background, with the mutation sites marked in red. b) Showed a
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