Bioscience Methods 2024, Vol.15, No.4, 184-195 http://bioscipublisher.com/index.php/bm 1 90 polymerase chain reaction (PCR) and Southern blot analysis (Figure 3) (Sun et al., 2022). For example, the overexpression of the OsC1 gene in rice resulted in the development of transgenic lines exhibiting markedly elevated anthocyanin levels (Upadhyaya et al., 2021). Figure 3 De-novo design colored rice (Adopted from Sun et al., 2022) Image caption: (A) Schematic illustration of recombinant construct CSA. S1 is promoted by CaMV35S, A1 and C1 are driven by the promoter of OsGluA2, which is highly expressed in the endosperm. (B–F) The purple anthocyanins accumulated in CSA transgenic lines such as callus (B), leaf blade (C), apiculus (D), culm (E), and pericarp (F). NIP, Nipponbare. Bar = 5 m in B, D, E and F. Bar = 5 cm in C (Adopted from Sun et al., 2022) 6.3 Phenotypic changes and anthocyanin accumulation The overexpression of R2R3-MYB genes in transgenic plants frequently gives rise to discernible phenotypic alterations, including modifications in pigmentation resulting from elevated anthocyanin accumulation. In rice, the overexpression of OsC1 resulted in purple pigmentation in various tissues, including leaves and panicles, due to the up-regulation of late ABP genes (Upadhyaya et al., 2021). Similarly, transgenic Arabidopsis plants overexpressing OjMYB1 exhibited enhanced anthocyanin content and up-regulation of structural genes related to anthocyanin biosynthesis (Feng et al., 2018). These phenotypic alterations are indicative of the successful modulation of the anthocyanin biosynthesis pathway by the overexpressed R2R3-MYB genes. 6.4 Comparative analysis with CRISPR/Cas9 studies A comparative analysis between overexpression and CRISPR/Cas9 studies provides insights into the functional roles of R2R3-MYB genes. While overexpression studies enhance the expression of target genes, CRISPR/Cas9 technology allows for precise gene editing, including knockout and knock-in mutations. For example, the overexpression of OsMYB3 in rice has been shown to result in increased anthocyanin production in the rice pericarp. Conversely, the CRISPR/Cas9-mediated knockout of specific R2R3-MYB genes could prove invaluable in identifying their contributions to the anthocyanin pathway (Figure 2) (Zheng et al., 2021). Such comparative studies are of great importance for the comprehension of the regulatory networks and potential redundancies among R2R3-MYB genes involved in anthocyanin biosynthesis. 7 Regulatory Mechanisms of R2R3-MYB Genes in Anthocyanin Pathway 7.1 Transcriptional regulation and promoter analysis The transcriptional regulation of R2R3-MYB genes in the anthocyanin pathway is a complex process, the outcome of which is influenced by several environmental and internal factors. R2R3-MYB TFs play a pivotal role in
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