黄连转录因子CcbHLH128的克隆、亚细胞定位及功能分析OA

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.