Genome-wide identification of the Dof Family in Dendrobium officinale and functional characterization of DoDof7 in enhancing polysaccharides biosynthesis during combined heat-drought stress

Dendrobium officinale possesses significant medicinal value, with polysaccharides as its primary bioactive compounds. Dof transcription factors play critical roles in regulating plant growth, development, metabolism, and responses to abiotic stress. However, comprehensive studies on the Dof gene family in D. officinale remain limited, and the functional roles and molecular mechanisms of its members are not yet fully understood. A genome-wide analysis identified 40 Dof family members, which were characterized in terms of gene structure, protein properties, chromosomal distribution, conserved motifs, phylogenetic relationships, and expression profiles. Promoter cis-element analysis revealed that many DoDof genes contain regulatory elements associated with stress responses, suggesting their involvement in abiotic stress adaptation. Expression profiling showed that DoDof7 is significantly upregulated under heat and drought stress. Subcellular localization confirmed its nuclear presence, and transcriptional activity assays affirmed its role as a transcription factor. Dual-luciferase reporter assays and electrophoretic mobility shift assays demonstrated that DoDof7 specifically binds to the promoter of DoGMPP2 , enhancing its expression. Functional analyses revealed that DoDof7 promotes polysaccharide biosynthesis by regulating DoGMPP2 expression and significantly increasing the mannose content in polysaccharide components. This study provides a comprehensive analysis of DoDof transcription factors in D. officinale , elucidating the molecular mechanism by which DoDof7 mediates heat and drought stress responses and regulates polysaccharide biosynthesis via DoGMPP2. These findings establish a theoretical framework and highlight potential molecular targets for enhancing stress resistance and bioactive compound content in medicinal plants through genetic engineering. • 40 DoDofs were identified in the D. officinale . • 10 DoDofs in the stem were differentially regulated under combined heat and drought stress, DoDof7 and DoDof26 were markedly upregulated. • DoDof7 was demonstrated to specifically bind to the DoGMPP2 promoter and activate its transcription. • Overexpression of DoDof7 enhances stem polysaccharide biosynthesis and increases the mannose content in the polysaccharide fraction.