Identification of DnaJ gene family in Ziziphus jujuba and expression analysis in response to drought stress

【Objective】Wild jujube (Ziziphus jujuba var. spinosa) is a variety of jujube (Ziziphus jujuba Mill). in the the Ziziphus genus of the Rhamnaceae family. It is a deciduous shrub characterised by remarkable drought tolerance and significant ecological value. The DnaJ transcription factor plays a crucial role in plants, frequently acting as a chaperone protein involved in abiotic stress tolerance. Based on the whole-genome identification of the DnaJ gene family in Z. jujuba var. spinosa, this study aimed to screen the key candidate genes within this gene family that respond to drought stress, and reveal the function and expression of the DnaJ gene family during the development of Z. jujuba var. spinosa. It would provide a theoretical reference for the systematic study of its drought resistance mechanisms and offer resources for optimizing elite drought- resistant varieties and breeding new cultivars in jujube. 【Methods】This study established a pot- based drought stress experiment with four treatment levels: well-watered (CK, control), mild drought (D1), moderate drought (D2), and severe drought stress (D3). The corresponding soil relative water contents were maintained at 70%-75%, 50%-55%, 35%-40%, and 25%-30%, respectively. The stress treatments were sustained for 20 days, then the roots, stems, and leaves of the drought-stressed jujube plants were collected for transcriptome sequencing. Based on the whole-genome data of the wild jujube, the members of the DnaJ transcription factor gene family were identified. Physicochemical properties were predicted using the online tool WoLF PSORT, while conserved motifs, domains, and gene structures were analyzed using TBtools, the MEME suite, and NCBI. Phylogenetic analysis was performed with MEGA11, and cis-acting elements were predicted using online tool PlantCARE. By integrating transcriptome data under drought stress, differential expression patterns of DnaJ genes of the various wild jujube organs were examined. The transcriptome results were further validated through qRT-PCR, enabling the screening of key drought-responsive genes within the ZjsDnaJ gene family. The principles for determining physiological indicators were as follows: soluble sugar content was measured using the anthrone colorimetric method; peroxidase (POD) activity was determined by the guaiacol method; catalase (CAT) activity was assessed via ultraviolet absorption; malondialdehyde (MDA) content was quantified using the thiobarbituric acid method; and proline (PRO) content was measured by the acid ninhydrin assay.【Results】A total of 23 ZjsDnaJ gene family members were identified in the whole genome of Z. jujuba var. spinosa. The expansion of the DnaJ gene family in the wild jujube was relatively conserved, which might be associated with its ecological adaptability. The physicochemical characterization revealed that the proteins encoded by these 23 ZjsDnaJ genes were localized in distinct subcellular compartments: cytoplasm (3), nucleus (9), mitochondria (1), and chloroplasts (10). These proteins included both acidic and alkaline types and were all hydrophilic, suggesting that their functional activities might primarily occur in acidic or alkaline solutions within these organelles, involving interactions with water molecules or other hydrophilic ions. Within the ZjsDnaJ gene family, 10 motifs were identified. All ZjsDnaJ genes contained the conserved Motif1 and Motif2, indicating a certain degree of conservation within the family. Additionally, eight conserved domains were detected, with the DnaJ domain being the most frequent while others served as auxiliary domains. This domain architecture suggested functional diversification during evolution. Furthermore, variation in gene structure, including differences in exon-intron organization among members, reflected functional diversity and evolutionary adaptation within the ZjsDnaJ family. The phylogenetic analysis of DnaJ genes from Z. jujuba var. spinosa, Arabidopsis thaliana, Solanum tuberosum, Solanum lycopersicum, and Z. jujuba cv.‘Dongzao’classified 162 genes into four distinct subgroups (GroupⅠ-Ⅳ), revealing both homologous relationships and genetic differentiation among species. The 23 ZjsDnaJ genes were distributed across 10 chromosomes, indicating a broad genomic distribution and suggesting that functional differentiation and coordinated regulation might enhance environmental adaptability. Cisacting elements regulating spatiotemporal specificity and expression levels, were investigated in ZjsDnaJ promoter regions. Thirty types of cis-acting elements associated with growth and development, environmental stress response, hormone response, and light response were identified, indicating the family's involvement in these physiological processes. Notably, abscisic acid-responsive elements (ABRE), MYB binding sites (MBS), and anaerobic response elements (ARE) might play crucial roles in drought stress response. The transcriptome analysis of the different organs under drought stress showed differential expression patterns of the ZjsDnaJ genes. Specifically, the ZjsDnaJ2, ZjsDnaJ3, ZjsDnaJ12, ZjsDnaJ14, ZjsDnaJ15, and ZjsDnaJ19 demonstrated high expression levels in the roots, stems, and leaves, respectively. Physiological indicators of drought tolerance, including malondialdehyde (MDA), proline, and soluble sugar content, as well as activities of antioxidant enzymes POD and SOD, were measured. Under drought stress, the leaves of the wild jujube exhibited increased levels of MDA, proline, and soluble sugar, along with enhanced POD and SOD activities, reflecting the plant's physiological adaptation to drought conditions.【Conclusion】The ZjsDnaJ gene family exhibited structural conservation and functional diversity, and would play a significant role in the drought stress response of Z. jujuba var. spinosa. Among its members, the ZjsDnaJ2, ZjsDnaJ3, ZjsDnaJ12, ZjsDnaJ14, ZjsDnaJ15, and ZjsDnaJ19 had been identified as key candidate genes mediating drought stress response. This study elucidated the expression profiles of the ZjsDnaJ gene family under drought stress in wild jujube, screened drought-resistant candidate genes, and might provide a foundation for breeding superior drought-tolerant varieties by offering critical genetic targets.