Maize Genomics and Genetics 2025, Vol.16, No.4, 202-218 http://cropscipublisher.com/index.php/mgg 212 various organs in space but also continuous defense at each stage in time, jointly ensuring the survival and growth of plants in high-temperature adverse conditions (Chen et al., 2024). 6 Candidate Gene Identification and Application Prospects 6.1 Identification and functional annotation of significantly differentially expressed genes under heat stress With the aid of transcriptomics technology, a large number of maize genes (DEGs) that were significantly differentially expressed under high-temperature stress were identified. These DEGs constitute the main gene pool of maize's heat response and also the starting point for exploring heat-tolerant functional genes. In the transcriptome analysis of corn seedlings or flowering tissues, it is often possible to detect that thousands of genes are upregulated or downregulated due to high-temperature treatment. For instance, Qian et al. (2019) reported that under short-term high temperature (42 ℃ treatment for 6 hours) during the seedling stage of corn, approximately 2 000 genes were significantly upregulated and about 1 500 genes were significantly downregulated, involving multiple functional categories such as heat shock proteins, antioxidant enzymes, osmotic regulatory substances, and signal proteins. Through bioinformatics annotation, these DEGs can be classified into several key pathways. Some upregulated DEGs belong to the "protein folding and repair" pathway (rich in HSP genes), some belong to the "reactive oxygen species scavenging" pathway (rich in SOD, APX and other genes), and some are involved in "hormone signaling" and "secondary metabolism", etc. For instance, the 142 core heat-stress response genes identified by Tang et al. (2023) were enriched in GO categories such as protein folding, ROS metabolism, and carbohydrate metabolism, indicating that these processes are significant in the heat tolerance of maize. Functional annotation and enrichment analysis of differentially expressed genes not only help identify known heat-resistant pathways (such as HSP, ABA, Ca2+ signaling, etc.), but may also discover new pathways that have not received attention before. For instance, some studies have found that the gene expression of the aquaporin family in corn also changes at high temperatures, suggesting that water transport may also be regulated under heat stress (He et al., 2022). For instance, some secondary metabolism-related genes (such as flavonoid synthase) are upregulated at high temperatures and may play a role in membrane stabilization or antioxidation. By conducting association analysis between DEGs and known heat-resistant QTLS, the range of candidate genes can be further narrowed down. Tang et al. (2023) compared the transcriptomes of heat-tolerant and sensitive maize, overlapping the common DEGs of both with heat-tolerant QTLS in maize yield and flowering period, and identified 42 differential genes located in the QTL region, such as genes encoding heat shock proteins, transcription factors, and cytoprotective enzymes. These genes are very likely the factors that affect the heat tolerance of corn. High-temperature induced DEGs identification provides a rich gene resource bank for the study of the heat tolerance mechanism of maize. Among these genes, there are many key genes that have been verified by existing research (such as ZmHSF, ZmHSP, ZmCAT1, etc.), as well as many new genes with unknown functions. Further functional annotation and cluster analysis of these candidate genes can serve as a basis for selecting important candidate genes. For instance, focus on screening genes that are specifically upregulated in heat-resistant varieties but do not increase in sensitive varieties, as well as genes that remain highly expressed at all stages of heat treatment. Such genes are more likely to be directly related to heat-resistant phenotypes. Next, functional experiments (such as genetic complementarity, gene editing, etc.) need to be conducted to verify its biological effects and select the genes that truly possess heat-resistant functions from them. 6.2 Promoter characteristics and regulatory patterns of heat-tolerant candidate genes For the candidate genes for corn heat tolerance obtained through screening, it is necessary to conduct in-depth research on their expression regulatory mechanisms, among which the cis-acting elements of the promoter and the upstream regulatory factors are the key entry points. The promoter regions of many heat-tolerant related genes are rich in typical thermal response elements (such as HSE, ABRE, DRE, etc.), revealing their transcriptional regulatory patterns. For instance, most HSP gene promoters have repetitive sequences of the thermal shock element HSE. The HSF protein can bind to HSE at high temperatures, thereby activating the transcription of the HSP gene. Experiments have demonstrated that the absence of HSE in the ZmHSP17.4 promoter significantly reduces its heat-induced expression level (Yao et al., 2019). For instance, the promoters of some heat-resistant antioxidant enzyme genes (such as ZmCAT1) contain ABA-responsive elements ABRE and DRE/CRT elements,
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