Wu Jing, Wang Shuting, Xing Shihai, Peng Daiyin
College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China.
MOE-Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials, Hefei 230012, China.
Biomolecules. 2025 Aug 29;15(9):1251. doi: 10.3390/biom15091251.
, 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 .
[一种濒危兰花物种]因其茎中具有巨大药理意义的多糖而闻名。磷酸甘露糖变位酶（PMM）对多糖积累起关键调节作用。[该物种]的转录调控仍知之甚少。本研究采用酵母单杂交（Y1H）系统来鉴定[该物种]的上游调控因子。克隆了2.15 kb的[该物种]启动子，揭示了多个应激和激素响应顺式元件（如ABRE、MYC、ERF）。构建了一个高复杂性Y1H文库（3.60×10 CFU），插入片段大小平均为1 - 2 kb。使用抗金担子素A（AbA）的Y1HGold [pAbAi - ]进行筛选，鉴定出11个候选克隆，包括四个转录因子家族（DOF、NAC、ERF、BES1）。通过成对Y1H显示抗AbA生长以及双荧光素酶测定证明[转录激活]，严格确认了相互作用。这代表了首个针对[该物种]的功能性cDNA文库资源以及与[该物种]相互作用的转录因子的鉴定。属于DOF、NAC、ERF和BES1家族的转录因子作为[该物种]调控因子的发现，阐明了多糖生物合成的转录网络。这为[该物种]中多糖生物合成工程建立了一个转录框架。
