Computational Molecular Biology 2025, Vol.15, No.2, 65-74 http://bioscipublisher.com/index.php/cmb 69 Interestingly, the regulatory methods of such genes are rather complex. Take the homologous gene BnaPAP2.A7 of BnMYB111 for example. Different splicing variants can have completely opposite effects on anthocyanin synthesis, which also indicates that we cannot make a blanket statement. Later, some people examined the metabolomics and transcriptomic data together and found that the accumulation patterns of anthocyanins and related flavonoids in petals of different colors were indeed different, and it was precisely the differential expression of genes such as BnMYB111 behind this. So, although color is just an appearance, the regulatory network hidden inside is much more intricate than we think. 4.2 Functional validation of BnMYB111 in regulating anthocyanin biosynthesis Recently, people have been paying particular attention to the BnMYB111 gene, mainly to figure out exactly how it regulates anthocyanin synthesis in rapeseed. As a member of the MYB transcription factor family, BnMYB111 is not alone-it often works together with "relatives" such as BnMYB90 and BnMYB114 to affect the expression of key anthocyanin synthesis genes like DFR and F3H (Chen et al., 2021). It was also found during the actual experiment that once the plants were overexpressed with BnMYB111, the anthocyanins in the petals increased significantly. No problem, it is a positive regulatory factor. Interestingly, not only rapeseed itself has this mechanism, but also in Arabidopsis thaliana, knocking out certain negative regulatory factors-such as AtMYB60, AtCPC or AtMYBL2-can deepen the coloring of anthocyanins. This can't help but make people wonder if there is a similar "brake gene" hidden in rapeseed, but we just haven't fully discovered it yet. 4.3 Experimental evidence: trANScriptomics and metabolomics analysis Through transcriptome and metabolome analysis, we now have a more specific understanding of the regulatory mechanism of rapeseed anthocyanin synthesis. For instance, the results of RNA-seq and metabolic profiling showed that genes such as ANS, DFR and UF3GT had significantly higher expression levels in red petals than in white and yellow petals, indicating that they were indeed directly related to red pigment formation. In fact, not only structural genes but also alleles play a prominent role. For instance, the type BnaA07.PAP2In-184-317, once introduced, can widely activate multiple genes along the anthocyanin synthesis pathway, turning the originally yellow flower into an apricot color and significantly increasing its accumulation. However, color formation does not rely on just one pathway. Transcriptional data also show significant differences in the expression of carotenoid and flavonoid related genes, indicating that the overall regulatory network is more complex than imagined (Zhang et al., 2020). These omics pieces of evidence, when put together, gradually pieced together the regulatory puzzle behind the patterns. 4.4 Implications for breeding and genetic engineering Understanding how anthocyanin synthesis in rapeseed is regulated is actually quite useful for both breeding and genetic engineering. The discovery of key genes such as BnMYB111, BnaA07.PAP2 and BnaA03.ANS has provided many clues for the future breeding of new varieties with brighter colors. These genes themselves can also be used as targets for genome editing, and perhaps some rapeseed with special flower colors and better resistance to environmental pressure can be produced. In practice, whether it is overexpression or gene knockout, it has been verified that they can indeed change the accumulation level of anthocyanins-in this way, not only is it good-looking, but perhaps it can also enhance some practicality in agriculture. In addition, the transcriptome and metabolome data are becoming increasingly abundant nowadays. There is more basis for selecting candidate genes, and it should be much smoother to cultivate what ideal traits in the future. 5 Evolutionary and Comparative Genomics Perspective 5.1 Evolutionary conservation of pigmentation pathways in Brassica species In fact, if you take a closer look at several cruciferous plants, you will find that the pathways through which they synthesize pigments are quite similar-whether it's carotenoids or anthocyanins, the functions of many key genes remain quite consistent. Take BnaA09.ZEP and BnaC09.ZEP in rape for example. Both of these genes encode zeaxanthin cyclooxygenase and are basically expressed only in flowers. Functionally, they can almost replace each other. It is obvious that they are quite important in flower color formation. This pattern seems to be quite common in other cruciferous plants as well. The situation is similar for anthocyanins. Regulatory genes like
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