Genomics and Applied Biology 2024, Vol.15, No.3, 120-131 http://bioscipublisher.com/index.php/gab 125 The integrated regulatory networks of miRNAs and transcriptome in rice response to cold stress involve specific miRNA-target interactions, network motifs and modules, and comparative analysis across different rice varieties. These networks play crucial roles in modulating gene expression and enhancing cold tolerance through complex regulatory mechanisms. 7 Experimental Approaches for Network Analysis 7.1 High-throughput sequencing technologies High-throughput sequencing technologies have revolutionized the study of gene regulatory networks by enabling comprehensive profiling of various molecular components. Next-generation sequencing (NGS) technologies, such as RNA-Seq and sRNA-Seq, allow for the detection of weakly expressed transcripts and the identification of regulatory RNAs, including miRNAs (Mazurier et al., 2022). These technologies facilitate the construction of integrated regulatory networks by providing detailed expression profiles of miRNAs and their target genes under different stress conditions (Liu et al., 2020; Mazurier et al., 2022). For instance, in Pisum sativum, sRNA-Seq and RNA-Seq were used to identify miRNA/mRNA target pairs involved in cold response, highlighting the role of miRNAs in antioxidative and multi-stress defense mechanisms (Mazurier et al., 2022). 7.2 Functional genomics tools Functional genomics tools are essential for elucidating the roles of miRNAs within gene regulatory networks. These tools include bioinformatics pipelines, high-throughput target identification platforms, and functional target validation methodologies (Steinkraus et al., 2016). For example, the integration of mRNA and miRNA expression data can reveal the regulatory control exerted by miRNAs on their target genes, as demonstrated in studies on mouse liver gene regulatory networks (Su et al., 2011). Additionally, the development of protocols for systematic assembling and integration of genomics data, such as miRNA-target interactions (MTIs), enables the construction of comprehensive miRNA regulatory maps (Zorc and Kunej, 2015). These integrative approaches help in understanding the complex interactions and regulatory mechanisms at play in response to environmental stresses. 7.3 Integrative multi-omics strategies Integrative multi-omics strategies combine data from various omics layers, such as genomics, transcriptomics, proteomics, and metabolomics, to provide a holistic view of the molecular networks involved in stress responses (Qin et al., 2016; Yang et al., 2021). These strategies involve the use of bioinformatics tools to integrate multi-dimensional data, generating novel insights into system-level gene regulations (Qin et al., 2016). For instance, multi-omics approaches have been applied to study the regulatory networks in crops like rice, elucidating the relationships between genomes and phenotypes under specific environmental conditions (Yang et al., 2021). The integration of different omics data layers allows for the construction of models that predict complex traits and enhance our understanding of molecular regulator networks for crop improvement (Wörheide et al., 2021; Yang et al., 2021). The combination of high-throughput sequencing technologies, functional genomics tools, and integrative multi-omics strategies provides a powerful framework for analyzing the integrated regulatory networks of miRNAs and the transcriptome in rice response to cold stress. These approaches enable the identification of key regulatory elements and their interactions, facilitating the development of targeted breeding programs to improve crop resilience and productivity. 8 Applications of Integrated Regulatory Networks 8.1 Implications for breeding cold-tolerant rice varieties The integration of miRNA and transcriptome sequencing has revealed significant insights into the regulatory mechanisms underlying cold stress tolerance in rice. For instance, the study on the chromosome single segment substitution line (CSSL) DC90, which exhibits a chilling tolerance phenotype, identified differentially expressed miRNAs (DEMs) and their target genes that are involved in various biological processes such as protein biosynthesis, redox processes, and chloroplast development (Zhao et al., 2022). This knowledge can be leveraged in breeding programs to select for rice varieties with enhanced cold tolerance by targeting specific miRNAs and their regulatory networks.
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