Computational Molecular Biology 2025, Vol.15, No.1, 38-52 http://bioscipublisher.com/index.php/cmb 48 the driving forces behind the genome, influencing drought resistance by regulating mRNA. The current breeding strategy is relatively clear: on the one hand, traditional methods are used to screen for superior stress-resistant traits; on the other hand, gene editing is employed to precisely regulate key transcription factors and lncrnas. However, in actual operation, it should be noted that the genetic background of different varieties varies greatly, and the same genetic modification may have completely different effects. Translating these molecular discoveries into field manifestations still requires a large number of experiments, but at least it provides new ideas for dealing with increasingly frequent droughts. 9 Current Advances in Genetic Engineering for Transcriptional Modulation 9.1 CRISPR-Cas9 applications in transcription factor editing The CRISPR-Cas9 gene editing technology is revolutionizing the way rapeseed varieties are improved. Taking glyphosate resistance as an example (Wang et al., 2021), researchers used a twin virus system to deliver CRISPR components and precisely modify the EPSPS gene to cultivate herbicide-resistant rapeseed seedlings. The most remarkable aspect of this technology lies in its ability to simultaneously handle multiple similar genes in the tetraploid genome of rapeseed (Li et al., 2018b), which is difficult to achieve with ordinary methods. Nowadays, this system is often used to edit the transcription factors that regulate stress resistance (Jaganathan et al., 2018), compared with traditional breeding, the target traits are obtained more quickly. However, in actual operation, it was also found that although CRISPR has high efficiency, the editing effects of different targets vary greatly (Tian et al., 2022), which reminds that multiple target schemes should be prepared when designing experiments. Overall, this technology has brought the improvement of rapeseed varieties into a new stage of precise regulation. From disease and drought resistance to increasing yields, the improvement of various agronomic traits has become easier to achieve. 9.2 Overexpression and knockdown approaches in stress tolerance In the research on the stress resistance of rapeseed, scientists often play the "addition and subtraction game"-either increasing the expression levels of key transcription factors or knocking out the genes that hold them back. For example, the bZIP transcription factor BnaABF2 (Zhao et al., 2016), when several copies are stuffed into Arabidopsis thaliana, the plants immediately become drought-tolerant and salt-tolerant because it can activate drought-resistant genes such as RD29B. Recently, it has become more popular to use CRISPR for "subtraction" (Sathee et al., 2022), directly knocking out the "brake pad" gene in the nutrient signaling pathway to enable crops to absorb nutrients more efficiently. However, it has been found in practice that regulating these genes requires moderation-overexpression of certain transcription factors can actually inhibit growth (Debbarma et al., 2019), just as overstepping the accelerator can cause problems. With the continuous emergence of new technologies (Nascimento et al., 2023), it is now possible to adjust these "switches" more precisely, allowing rapeseed to maintain good growth in harsh environments. The principle is simple, yet it offers unlimited possibilities for cultivating stress-resistant varieties. 9.3 Future directions for genetic modulation in rapeseed There are several particularly notable directions for future research on enhancing the stress resistance of rapeseed. There are more and more new applications of CRISPR technology-now not only gene knockout can be performed, but also the expression level of genes can be precisely regulated by CRISPRa and CRISPRi, just like installing adjustable knobs on genes. Even more remarksable is the multi-editing technology (Debbarma et al., 2019), which can adjust multiple genes at one time, enabling rapeseed to possess multiple abilities such as drought resistance and salt-alkali tolerance simultaneously. However, what is most anticipated are still those new transcription factors that have not yet been discovered (Jain, 2015), and each one found may bring about new breeding ideas. Combine these technologies (Bortesi and Fischer, 2015), it is possible to cultivate more "hardy" rapeseed varieties, which not only have stable yields but also can cope with increasingly extreme climates. Of course, ultimately these studies must return to the fields and truly help farmers solve practical problems. This is where the value of agricultural research lies.
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