Computational Molecular Biology 2025, Vol.15, No.5, 227-234 http://bioscipublisher.com/index.php/cmb 228 to deepen our understanding of the molecular mechanism of drought tolerance in corn and provide valuable genetic resources for breeding programs aimed at enhancing the drought resistance of corn. 2 Biological Characteristics and Regulatory Mechanisms of miRNAs 2.1 Biogenesis pathways and classification of miRNAs miRNA may sound unremarkable, but in fact, it often plays a key role in regulating gene expression within plants. miRNA, which is usually only 20 to 24 nucleotides in length, is an endogenous non-coding RNA. Generally, it does not directly encode proteins, but it has a significant impact on the "switch" of gene expression. Its generation begins when the MIR gene is transcribed into pri-miRNA by RNA polymerase II, and this transcript has a typical stem-loop structure. Then, these PRI-mirnas will be processed in the cell nucleus by Dicer-like proteins, mainly DCL1, cleaved into pre-mirnas, and gradually transformed into double-stranded mature mirnas. Afterwards, miRNA binds to the AGO protein to assemble into a RISC complex, which acquires the ability to "quiet certain genes" - achieved by cutting mRNA or preventing it from being translated into proteins (Song et al., 2019; Wang et al., 2019; Zhan and Meyers, 2022). Of course, not all mirnas are exactly the same; their conservation levels, precursor structures, and processing methods vary significantly. Some miRNA families can be found in multiple species, while others only appear in specific groups. 2.2 Regulatory patterns of miRNAs in response to drought stress in plants When drought occurs, the expression pattern of miRNA within plants is quite different. Behind this change lies the process of plants' self-adjustment. Not all mirnas are involved, but some mirnas, such as miR159, miR169, and miR393, have been repeatedly demonstrated to be involved in pathways such as ABA signaling, oxidative stress defense, and root regulation (Islam et al., 2022). These mirnas target the transcription factors or key signaling molecules that control the stress response and adjust the "neural response speed" of the entire system by adjusting up and down. However, such regulation is not uniform. The expression intensity and mode of miRNA may vary significantly across different tissues, varieties, and even at different growth stages. Nowadays, with the help of high-throughput sequencing technology, researchers have identified many mirnas in staple food crops such as corn and wheat that are induced or inhibited by drought, and their "node" status in the regulatory network is becoming increasingly clear. 2.3 Interactions between miRNAs and target genes and their modulation of signaling pathways How exactly does miRNA shut up a gene? It relies on "matching oneself". Once it finds a complementary mRNA sequence to itself, it can bind to it, and then trigger mRNA degradation or at least prevent it from being translated. In plants, mirnas most frequently target various transcription factors, such as the "familiar faces" like MYB, NF-YA, SPL, ARF, and WRKY (Samad et al., 2017). These transcription factors themselves control a bunch of other genes. Therefore, when miRNA takes action, it is equivalent to influencing an entire series of signaling responses, such as ABA signaling, auxin regulation and ROS clearance mechanism (Sharma et al., 2025). What's more interesting is that miRNA and target genes usually show a state of "one rising and the other falling", where one side is elevated while the other is often suppressed. This reverse expression can achieve very fine regulation. Nowadays, by means such as degradation omics sequencing and RACE-PCR, researchers can relatively clearly confirm the relationship between these mirnas and their targets, and some have even become potential tools for studying drought-resistant breeding. 3 Application of Deep Sequencing in miRNA Identification 3.1 Overview of sRNA sequencing platforms and experimental procedures Not everyone can realize at the beginning how much convenience the popularization of small RNA (sRNA) sequencing has brought to the research of plant miRNA. High-throughput platforms like Illumina HiSeq and NextSeq, although they may sound more technical, have actually become the "standard equipment" for analyzing miRNA. Take corn as an example. Researchers usually extract total RNA from the tissues of the control group and the drought treatment group, and then carry out a series of operations: screening out fragments of 18 to 32 nucleotides in length, adding linkers, reverse transcription, PCR amplification, and finally sending it for on-machine sequencing (Jiao et al., 2022; Cheng and Wang, 2025). Of course, setting up more time points and
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