Construction of Yeast One-Hybrid Library of and Screening of Potential Transcription Factors Regulating Gene Expression.

, an endangered orchid species, is renowned for its polysaccharides with vast pharmacological significance in stems. Phosphomannomutase (PMM) critically regulates polysaccharide accumulation. Transcriptional regulation of remains poorly characterized. This study employed a yeast one-hybrid (Y1H) system to identify upstream regulators of . The 2.15 kb promoter was cloned, revealing multiple stress- and hormone-responsive cis-elements (e.g., ABRE, MYC, ERF). A high-complexity Y1H library (3.60 × 10 CFU) was constructed with insert sizes averaging 1-2 kb. Screening using aureobasidin A (AbA)-resistant Y1HGold [pAbAi-] 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 activation. This represents the first functional cDNA library resource for and identification of TFs interacting with . The discovery of TFs belonging to DOF, NAC, ERF, and BES1 families as regulators elucidated the transcriptional network underlying polysaccharide biosynthesis. This establishes a transcriptional framework for engineering polysaccharide biosynthesis in .