Genome-Wide Identification of the DREB Gene Family in Juglans regia and Transcriptome Analysis under Stress Conditions

Objective To elucidate the molecular mechanism of Juglans regia in response to drought stress, identify members of its DREB gene family, and analyze their expression patterns under abiotic stress.

Method Members of the DREB family were identified from the walnut genome, and phylogenetic trees were constructed using MEGA. Conserved motifs, domains, and physicochemical properties of proteins were analyzed via tools such as MEME and NCBI CD-Search. Chromosomal localization and collinearity analysis were performed using TBTools. Walnut seedlings were subjected to PEG6000-simulated drought and heat stress treatments; differentially expressed genes (DEGs) were screened using RNA-seq (with DESeq package, criteria: p<0.05 and |log₂FoldChange|≥2) and validated by RT-qPCR. Cis-acting elements in the promoters of hub genes were predicted using PlantCARE.

Result A total of 55 DREB family members were identified, clustered into 6 groups. The proteins had 113 - 463 amino acids, molecular weights of 13220.24 - 51862.71 Da, isoelectric points of 4.72 - 11.2; 98.2% had an instability index >40, and all were hydrophobic. Genes were mostly distributed at chromosome ends. Under drought stress, 10 DEGs (7 up-regulated, 3 down-regulated) were found in roots, and 15 DEGs (11 up-regulated, 4 down-regulated) in leaves. Seven hub genes were differentially expressed under both stresses. RT-qPCR confirmed the accuracy of RNA-seq results. Promoters of hub genes contained elements related to light response, ABA response, drought induction, and low-temperature response.

Conclusion Members of the walnut DREB family are predominantly positively regulated under abiotic stress; intron-less or few-intron features may enhance their stress response efficiency. Some hub genes (e.g., DREB1A, DREB1B) play key roles in low-temperature and drought stress. This study provides important genetic resources and theoretical basis for stress-resistant molecular breeding of walnuts.