Identification of the ZmDUF966 Gene Family in Maize, Analysis of Its Expression Under Cold Stress, and Preliminary Investigation of the ZmDUF966-10 Regulatory Network

Maize (<i>Zea mays</i> L.) originated in tropical and subtropical regions. During its growth and development, cold stress severely threatens seedling survival rates and final yield by inducing oxidative stress, compromising cell membrane integrity, and causing "physiological drought." The Domain of Unknown Function 966 (<i>DUF966</i>) gene family comprises a class of regulatory factors containing conserved domains of undetermined function. Although they are considered to be extensively involved in plant growth, development, and stress response, their specific roles within the maize cold-tolerance regulatory network remain to be explored. In this study, 10 <i>ZmDUF966</i> family members were identified via genome-wide analysis, and their phylogenetic relationships, gene structures, conserved motifs, chromosomal localizations, and cis-acting elements were systematically analyzed. The results indicate that the <i>ZmDUF966</i> family is highly conserved among Poaceae species, and its promoters are enriched with stress-responsive elements such as LTR and ABRE. The core gene, <i>ZmDUF966-10</i>, was significantly up-regulated (approximately 35-fold at 48 h, <i>p</i> < 0.05) as validated by RT-qPCR under cold stress and is post-transcriptionally regulated by conserved miRNAs such as zma-miR159. Further yeast two-hybrid experiments revealed a preliminary physical interaction between the ZmDUF966-10 protein and an ABA/WDS-induced protein, suggesting its potential involvement in ABA-mediated stress signaling, though functional validation remains to be conducted. In conclusion, this study identifies <i>ZmDUF966-10</i> as a promising candidate gene that responds to cold signals through multi-level regulatory networks, providing a valuable gene resource for further functional characterization and potential application in cold-tolerant maize improvement.