Cotton Genomics and Genetics 2025, Vol.16, No.5, 232-240 http://cropscipublisher.com/index.php/cgg 235 dual-luciferase reporter assay. This idea is relatively straightforward: Insert circRNA and miRNA together into an expression vector and observe whether the reporter gene activity changes. If there is a change, it indicates that binding has occurred between them (Yang et al., 2020). Then comes RNA immunoprecipitation (RIP). This step is like "molecular-level net pulling", using antibodies against Argonaute protein to pull out the miRNA-circRNA complex, thereby determining whether they are "in the same frame" (Yang et al., 2022). Finally, there is the RNA pull-down experiment, which is more like the "fishing method"-using biotin-labeled probes to catch the circRNA or miRNA of interest and see what kind of "fish" are caught (Bai et al., 2018; Singh et al., 2023). These methods are often used in combination, one to verify the structure and the other to supplement the function, confirming from different aspects the true role of circRNA in the miRNA regulatory network. 4 Functional Roles of circRNAs in Cotton Development and Stress Responses 4.1 Regulation of fiber development and elongation via miRNA sequestration During the development of cotton fibers, circRNA is not an onlooker. Although experimental evidence regarding its direct involvement in such regulation is still accumulating at present, there are already many indirect clues suggesting that it may have been involved in this complex process. For instance, it can "intercept" specific mirnas through the ceRNA mechanism, reducing the inhibition of these mirnas on certain key mrnas, thereby influencing cell differentiation and growth. This mechanism has long been reported in other plants, and in cotton, circRNA shows obvious expression dynamics and stage specificity at different developmental stages, which further suggests that it is not insignificant. For instance, some studies have identified that certain circRNA-miRNA-mRNA networks have tissue-specific expression patterns during the cotton fiber formation period (Li et al., 2024). So, even though many details have not yet been fully revealed, the role of circRNA in fiber growth seems to be increasingly difficult to ignore. 4.2 circRNA-mediated modulation of abiotic stress responses (e.g., drought, salt) When plants encounter abiotic stresses such as drought or high salt content, their internal gene expression networks often adjust rapidly. Cotton is no exception. Some recent studies have shown that the expression of circRNA is not stable under these stress conditions and instead undergoes significant changes. In other words, their existence seems to be "related" to environmental changes. Take drought as an example. By using deep sequencing technology, scientists have discovered that some circrnas are involved in the ceRNA regulatory network of specific tissues and even specific varieties. This type of circRNA can adsorb miRNA, thereby affecting the expression of certain stress response genes (Yadav et al., 2024). A widely mentioned example is circ125 and miR7484b/miR7450b, which may have constructed a new regulatory relationship in the context of drought. This also indicates from one aspect that circRNA may play a certain role as a "regulator" in the adaptation to abiotic stress, and this role is highly context-specific and complex. 4.3 Role of circRNAs in disease resistance and defense signaling Similar to environmental stress, the invasion of pathogens can also trigger a series of molecular reactions within cotton. Interestingly, circRNA also "came to life" at this time. In a study on Fusarium wilt, researchers observed that hundreds or thousands of circrnas were differentially expressed in different cotton strains after infection (Xiang et al., 2018). Some circrnas are significantly activated in disease-resistant strains, but not so obviously in susceptible strains. What is more notable is that many of these circrnas are related to NBS genes, and the NBS gene family is precisely a core member of the plant immune response. This discovery suggests that circRNA may not directly interact with pathogens but indirectly affect the expression of downstream immune-related genes by regulating the activity of miRNA. This regulatory approach adds a new level to the plant's defense mechanism and also opens up a new breakthrough for enhancing the disease resistance of cotton. 5 High-Throughput Identification and Profiling of circRNAs in Cotton 5.1 RNA-seq and bioinformatic pipelines (e.g., CIRI, find_circ) When looking for circular RNAs in cotton, RNA sequencing is usually an indispensable step, especially for the version after the rRNA has been removed. However, light sequencing is not enough; some specialized bioinformatics tools are also needed to "dig out" circular RNAs from the data. Programs like CIRI and find_circ
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