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

Abstract

Dendrobium huoshanense, an endangered orchid species, is renowned for its polysaccharides with vast pharmacological significance in stems. Phosphomannomutase (PMM) critically regulates polysaccharide accumulation. Transcriptional regulation of DhPMM remains poorly characterized. This study employed a yeast one-hybrid (Y1H) system to identify upstream regulators of DhPMM. The 2.15 kb DhPMM 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-DhPMM] 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 DhPMM activation. This represents the first functional cDNA library resource for D. huoshanense and identification of TFs interacting with DhPMM. The discovery of TFs belonging to DOF, NAC, ERF, and BES1 families as DhPMM regulators elucidated the transcriptional network underlying polysaccharide biosynthesis. This establishes a transcriptional framework for engineering polysaccharide biosynthesis in D. huoshanense.