世界科学技术-中医药现代化2026，Vol.28Issue(3)：716-726,11.DOI:10.11842/wst.20251021008
黄连转录因子CcbHLH128的克隆、亚细胞定位及功能分析
Cloning,Subcellular Localization,and Functional Analysis of the Coptis chinensis Transcription Factor CcbHLH128
摘要
Abstract
Objective This study aims to clone the CcbHLH128 bHLH(basic helix-loop-helix)transcription factor from Coptis chinensis Franch.and investigate its role in benzylisoquinoline alkaloid biosynthesis.Methods CcbHLH128 was cloned from the C.chinensis genome database.Bioinformatics tools were used to analyse the amino acid sequence,predict physicochemical properties and identify structural domains.Phylogenetic analysis was performed using MEGA11.Tissue-specific expression patterns were examined via quantitative real-time PCR(qRT-PCR).Dual-luciferase assays were conducted to assess the transcriptional activation of key benzylisoquinoline alkaloid biosynthesis genes(CcCYP719A2,CcCYP80G2,CcNCS2,CcNCS5,CcNMCH and CcOMT4)by CcbHLH128.Results The CcbHLH128 gene was successfully cloned from C.chinensis and exhibited the highest expression levels in its fibrous roots.Its open reading frame(ORF)sequence spans 2199 bp,encoding 732 amino acids with a relative molecular mass of 81588.In the absence of transmembrane domains and signal peptides,it functions as a hydrophilic,unstable protein localised to the nucleus.Four secondary structures were identified,with disordered coils constituting the largest proportion at 61.20%.CcbHLH128 belongs to bHLH family ⅩⅢ subfamily,exhibiting significant transcriptional activation effects on CcCYP719A2,CcNCS2 and CcNMCH.Conclusion This study lays the foundation for further elucidating the role of bHLH-type transcription factors in the regulatory network governing benzylisoquinoline alkaloid synthesis in Coptis chinensis.关键词
黄连/bHLH转录因子/生物碱/亚细胞定位/基因表达/双荧光素酶报告基因实验Key words
Coptis chinensis Franch./bHLH transcription factor/Alkaloids/Subcellular localization/Gene expression/Dual-luciferase assays分类
医药卫生引用本文复制引用
李国凤,王雨婷,王瑾,田徐芳,李伟,张诗,刘迪,刘义飞..黄连转录因子CcbHLH128的克隆、亚细胞定位及功能分析[J].世界科学技术-中医药现代化,2026,28(3):716-726,11.基金项目
湖北时珍实验室首席科学家项目(HSL2024SX0006):黄连资源多组学评价与利用研究,负责人:刘义飞 （HSL2024SX0006）
湖北省科技厅湖北省自然科学基金面上项目(2024AFB956):响应JA信号的bHLH转录因子调控黄连生物碱合成的分子机制解析,负责人:刘迪. （2024AFB956）
