Bioscience Methods 2025, Vol.16, No.6, 280-288 http://bioscipublisher.com/index.php/bm 283 (especially at positions 10 and 11) can cleavage be initiated. The target site may occur in the coding region of mRNA or in the untranslated region. This almost exacting complementarity determines the precision of its regulation. However, there are exceptions - some studies have found that certain mirnas can recognize non-classical binding sites, which has led people to reconsider whether their regulatory scope is broader than previously thought. In addition to sequence complementarity, the structure of the target mRNA itself and the type of AGO protein can also affect the silencing efficiency (Liu et al., 2017; Ajila et al., 2023). 4 Expression Profiling of microRNAs in Rye under Drought Stress 4.1 Application of high-throughput sequencing in miRNA research To understand the expression status of miRNA in plants under stress conditions such as drought, relying solely on traditional methods is far from sufficient. High-throughput sequencing (HTS) technology, especially next-generation sequencing (NGS), has taken a dominant position in miRNA research precisely because it can rapidly scan and quantify mirnas across the entire genome. This type of technology can not only capture conserved mirnas but also discover new candidate molecules. Typically, the results of HTS are also combined with bioinformatics analysis, such as predicting the interaction relationship between miRNA and its target mRNA, and the subsequent verification work is generally accomplished by means such as qRT-PCR and RACE PCR (Zhakypbek et al., 2025). This "screening first, then verification" process has become a routine operation for studying the mechanism of miRNA's action in plant stress responses. 4.2 Identification of differentially expressed miRNAs induced by drought stress Not all mirnas respond to drought, but through HTS comparative analysis, a batch of "drought-sensitive" mirnas have indeed been found in many crops. In crops such as rice, wheat, barley and alfalfa, the expression levels of miR156, miR159, miR164, miR169, miR172, miR396 and miR398 have all changed under drought conditions, with some being activated and others suppressed. The specific situation also depends on the variety, tissue type and degree of stress (Ferdous et al., 2017; Qiu et al., 2020). Such expression differences often carry strong tissue specificity or genotype differences, which also indicates that these mirnas may play a relatively detailed role in drought adaptation. For rye, identifying these differentially expressed mirnas is undoubtedly a crucial step in understanding its drought resistance mechanism. 4.3 Screening and functional prediction of key drought-related miRNAs Identifying the candidate mirnas is just the starting point. The next key point is to figure out which genes they regulate and the roles of these genes in drought resistance. Through target gene prediction and functional enrichment analysis, it can be seen that many mirnas are related to ABA signaling, root development, antioxidant response, metabolic regulation and other links (Sharma et al., 2025). Regulatory pathways such as the effect of miR164 on NAC transcription factors, the regulation of MYB by miR159, and the regulation of NFYA by miR169 have all been widely mentioned in the context of drought stress (Liu et al., 2019; Zhakypbek et al., 2025). Many studies have further conducted transgenic or expression verification. For example, miR408, once overexpressed in ryegrass, not only improved the water state of the leaves, but also enhanced the antioxidant capacity (Hang et al., 2020). Although these results are not yet the end, they have already demonstrated that the value of miRNA as a breeding tool is becoming increasingly clear. 5 Functional Analysis of miRNA Target Genes in Rye 5.1 Target gene prediction and functional annotation Which targets exactly does miRNA regulate? This matter needs to be judged by some "accurate" tools. Methods such as psRNATarget, TargetFinder, and degradation omics sequencing are widely used in plant research, especially in finding mrnas complementary to miRNA sequences in rye or other cereals (Figure 2) (Tang et al., 2022). However, prediction alone is not enough. The key lies in what exactly these target genes have done. Very often, they encode transcription factors (such as NAC, MYB, SPL, etc.), signaling proteins, or enzymes involved in ABA signaling, ROS detoxification and root development. These functions are basically closely related to drought resistance (Aravind et al., 2017). Like miR156, miR164 and miR159, the corresponding targets are SPL, NAC and MYB genes respectively, and they are basically all "involved" in the stress pathway. Of course, these
RkJQdWJsaXNoZXIy MjQ4ODYzNA==