Construction of Yeast One-Hybrid Library of Dendrobium huoshanense and Screening of Potential Transcription Factors Regulating DhPMM Gene Expression

<i>Dendrobium huoshanense</i>, an endangered orchid species, is renowned for its polysaccharides with vast pharmacological significance in stems. Phosphomannomutase (PMM) critically regulates polysaccharide accumulation. Transcriptional regulation of <i>DhPMM</i> remains poorly characterized. This study employed a yeast one-hybrid (Y1H) system to identify upstream regulators of <i>DhPMM</i>. The 2.15 kb <i>DhPMM</i> promoter was cloned, revealing multiple stress- and hormone-responsive cis-elements (e.g., ABRE, MYC, ERF). A high-complexity Y1H library (3.60 × 10⁹ CFU) was constructed with insert sizes averaging 1-2 kb. Screening using aureobasidin A (AbA)-resistant Y1HGold [pAbAi-<i>DhPMM</i>] identified 11 candidate clones, including four transcription factor families (DOF, NAC, ERF, BES1). Interactions were rigorously confirmed by pairwise Y1H showing AbA-resistant growth and dual-luciferase assays demonstrating <i>DhPMM</i> activation. This represents the first functional cDNA library resource for <i>D. huoshanense</i> and identification of TFs interacting with <i>DhPMM</i>. The discovery of TFs belonging to DOF, NAC, ERF, and BES1 families as <i>DhPMM</i> regulators elucidated the transcriptional network underlying polysaccharide biosynthesis. This establishes a transcriptional framework for engineering polysaccharide biosynthesis in <i>D. huoshanense</i>.