Genomics and Applied Biology 2024, Vol.15, No.4, 200-211 http://bioscipublisher.com/index.php/gab 205 2) Gene expression analysis: Quantitative real-time PCR (qRT-PCR) can be employed to quantify the expression levels of R2R3-MYB genes and their target anthocyanin biosynthetic genes in diverse tissues and developmental stages of colored rice. This approach can facilitate the establishment of a correlation between gene expression and anthocyanin accumulation (Upadhyaya et al., 2021; Yang et al., 2023). 3) Protein-protein interaction studies: Yeast two-hybrid assays and co-immunoprecipitation can be employed to investigate interactions between R2R3-MYB proteins and other TFs, such as bHLH proteins, which are known to form complexes that regulate anthocyanin biosynthesis (Li et al., 2020; Kim et al., 2021). 4) Subcellular localization: Fluorescent protein tagging and confocal microscopy can be employed to ascertain the subcellular localization of R2R3-MYB proteins, thereby elucidating their functional roles as transcriptional regulators (Feng et al., 2018; Kim et al., 2021). 5) Metabolite analysis: High-performance liquid chromatography (HPLC) can be employed to quantify anthocyanin levels in transgenic and wild-type rice plants, thereby enabling an evaluation of the impact of R2R3-MYBgene manipulation on anthocyanin biosynthesis (Zhu et al., 2017; Upadhyaya et al., 2021). The functional roles of R2R3-MYB genes in anthocyanin biosynthesis in colored rice can be comprehensively characterized by employing these experimental approaches, thereby providing valuable insights into their potential applications in crop improvement and biofortification. 7 Integration of Phylogenetic and Expression Data 7.1 Correlation between phylogenetic clades and expression profiles Integrating phylogenetic analysis with gene expression data facilitates a comprehensive understanding of the functional dynamics of R2R3-MYB genes in colored rice. By correlating the phylogenetic clades with expression profiles obtained from various tissues and developmental stages, distinct expression patterns corresponding to specific clades were observed. Clades comprising genes with high sequence similarity often displayed comparable expression profiles, indicating the presence of conserved regulatory mechanisms. For example, genes within clade A were predominantly expressed in leaf tissues, indicating a potential role in photosynthesis-related processes. In contrast, genes in clade B exhibited high expression levels in root tissues, suggesting a potential role in root development and stress responses. This correlation highlights the evolutionary conservation of gene function within clades and demonstrates the value of the phylogenetic context in predicting gene expression patterns (Piao et al., 2019; Kang et al., 2022). 7.2 Insights into gene function from integrated analysis The integration of phylogenetic and expression data provides valuable insights into the functional roles of R2R3-MYBgenes, highlighting their biological significance. For example, genes in clade C, characterized by high expression during flower development, are likely involved in regulating floral organ differentiation and development. This hypothesis is supported by the presence of known floral regulators within this clade. Similarly, genes in clade D, with elevated expression under abiotic stress conditions, suggest roles in stress response pathways. The co-expression of R2R3-MYB genes with known stress-responsive genes further corroborates this functional association. By linking phylogenetic clades with specific expression profiles and functional annotations, potential roles for uncharacterized R2R3-MYB genes can be inferred, guiding future experimental validation and functional studies (Upadhyaya et al., 2021; Chen et al., 2022). 7.3 Case studies of key R2R3-MYBgenes To demonstrate the practical applications of our integrated analysis, we present case studies of key R2R3-MYB genes with significant functional implications. The gene OsMYB30, a member of the R2R3-MYB transcription factor family, plays a critical role in regulating the expression of phenylalanine ammonia-lyase (PAL) genes, which are essential for lignin biosynthesis and plant defense mechanisms. Prior research has demonstrated that the tissue-specific expression of PALis regulated by the R2R3 MYB transcription factor through binding to AC-rich elements. He et al. (2019) identified a mutant with
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