Rice Genomics and Genetics 2024, Vol.15, No.1, 28-35 http://cropscipublisher.com/index.php/rgg 29 Figure 1 DFOT divergence in rice subspecies Figure 2 through comparative transcriptomic analysis of lodicules between indica (TFB) and japonica (ZH11), revealed the gene expression differences at various time points. Figure 2a showed a Principal Component Analysis (PCA) highlighting the differences in the transcriptome datasets of TFB and ZH11 lodicules at different times. The time points T18 and Z18 represent 18:00 the day before floret opening, T9 and Z9 represent 9:00 (corresponding to 1 h and 3 h before opening for TFB and ZH11, respectively), Z11 represents 11:00 (1 h before ZH11 opening), and TF and ZF represent the peak of floret opening (approximately 10:00 for TFB and 12:00 for ZH11). The Venn diagram in figure 2b showed the number of upregulated differentially expressed genes (DEGs) in the comparison of Cluster18 genes with T9 versus Z9. Figure 2c displayed a Gene Ontology (GO) enrichment analysis of 251 intersecting genes, with Molecular Function (MF) and Biological Process (BP) indicated in different colors representing levels of P-value, with "transcription factor activity" highlighted in red. Figure 2d showed the expression analysis of 11 transcription factors selected through GO analysis, with the heatmap numbers representing average FPKM values. Figure 2e presented the RT-qPCR analysis of OsMYB8 expression levels in the lodicules of TFB and ZH11 at 9:00 AM in June and October 2021, with values representing mean ± standard error (SEM), n=3 biological replicates, and the significance at each time point evaluated by two-sided Student's t-test, providing P-values. Therefore, indicating significant differences in the expression regulation of lodicules between indica and japonica rice, especially in the regulation of flowering time, where the OsMYB8 gene showed differential expression, possibly contributing to the DFOT differences between the two subspecies. Figure 3 displayed the use of the CRISPR/Cas9 gene editing technique to create OsMYB8 mutants and analyzed their effects on the diurnal floret opening time (DFOT) in different backgrounds (ZH11 and TFB). Figure 3a showed the successful introduction of targeted mutations in the OsMYB8 gene using CRISPR/Cas9 technology. Figures 3b, e, j compared the inflorescences of ZH11 and Osmyb8ZH mutants (b), TFB and Osmyb8TF mutants (e), and ZH11 and OsMYB8TF/ZH11 transgenic lines (j) at different times. Figures 3c, f, k recorded the number of florets opened at different times in June, while figures 3d, g, l showed the number of florets opened on different days after flowering, both showing clear differences. Figure 3h presented the vector structure used to construct the OsMYB8TF/ZH11 transgenic plants. Figure 3i analyzed the expression levels of the OsMYB8 gene in the lodicules of OsMYB8TF/ZH11 transgenic lines by RT-qPCR, with ZH11 as a negative control, showing a significant increase in OsMYB8 expression in the transgenic lines, determined by two-sided Student’s t-test for significance.
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