南方农业学报2026，Vol.57Issue(3)：673-685,13.DOI:10.3969/j.issn.2095-1191.2026.03.006
齿瓣石斛黄酮醇合成酶基因(DdFLSs)克隆及体外功能分析
Cloning and in vitro functional analysis of flavonol synthase genes from Dendrobium devonianum(DdFLSs)
摘要
Abstract
[Objective]To clone the flavonol synthase(FLS)genes from Dendrobium devonianum(DdFLSs)and ana-lyze their in vitro functions,which could provide theoretical basis for further exploring the regulatory mechanism of flavo-noid biosynthesis in Dendrobium devonianum.[Method]Based on the transcriptome sequencing data of Dendrobium devo-nianum leaves,candidate DdFLSs genes were screened and cloned.Bioinformatics analysis and prokaryotic system expression were performed on the cloned DdFLS genes,and the catalytic activity of DdFLSs was verified by in vitro en-zyme activity assay.[Result]Two DdFLSs genes were successfully cloned and named DdFLS1 and DdFLS2 respectively.The open reading frames(ORF)of DdFLS1 and DdFLS2 genes were both 1002 bp in length,each encoding 333 amino acids,with relative molecular weights of 37.54 kD and 37.71 kD respectively.Both proteins were unstable hydrophilic proteins and contained typical conserved domains including 2OG-Fe(Ⅱ)-Oxy,PcbC,and PLN02704,belonging to the Fe2+and 2-oxoglutarate-dependent dioxygenase(2-ODD)superfamily.For the secondary structure of both proteins,α-helix and random coil accounted for a high proportion,and their tertiary structures shared 70％identity with the tem-plate B0FYE8.1.A Flavonol synthase.The amino acid sequence similarity of DdFLS1 and DdFLS2 with FLS proteins from Arabidopsis thaliana,Allium cepa and Dendrobium officinale ranged from 75.98％to 98.70％.Among them,DdFLS1 and DdFLS2 had the highest amino acid sequence similarity with FLS from Dendrobium officinale,which were in the same genus,and clustered in the same small branch in the phylogenetic tree,indicating the closest genetic relationship between them.Under the induction condition of 0.5 mmol/L isopropyl β-D-thiogalactoside(IPTG)at 16℃for 14 h,DdFLS1 and DdFLS2 proteins were successfully expressed in the prokaryotic expression system.The purification effect was the best when using 250 mmol/L imidazole elution buffer.In vitro enzyme activity verification showed that,DdFLS1 and DdFLS2 proteins did not exhibit typical FLS activity,that was,they could not catalyze dihydrokaempferol,dihydro-quercetin,and dihydromyricetin to generate corresponding flavonols.However,they could catalyze naringenin to gener-ate dihydrokaempferol,indicating that they possessed the activity of flavanone 3-hydroxylase(F3H).[Conclusion]Two DdFLSs genes have been cloned from Dendrobium devonianum,their encoded proteins do not have FLS activity but have F3H activity.This may be due to mutations in two key sites of DdFLS1 and DdFLS2 proteins,leading to the transforma-tion of their activities.关键词
齿瓣石斛/黄酮醇合成酶(FLS)/体外酶活/分子对接Key words
Dendrobium devonianum/flavonol synthase(FLS)/in vitro enzyme activity/molecular docking分类
农业科技引用本文复制引用
张娜,孙春梅,冉璜馨,常健红,姚爱芹,杨亦晨,唐军荣,韩庆莉..齿瓣石斛黄酮醇合成酶基因(DdFLSs)克隆及体外功能分析[J].南方农业学报,2026,57(3):673-685,13.基金项目
国家中药材产业技术体系项目(CARS-21) （CARS-21）
云南省科技厅乡村振兴科技专项(202404BP090087) （202404BP090087）
西南林业大学林学学科和重点实验室开放基金项目(LXXK-2024M14) China Agriculture Research System-Traditional Chinese Medicinal Herbs(CARS-21) （LXXK-2024M14）
Rural Re-vitalization Science and Technology Special Project of Yunnan Science and Technology Department(202404BP090087) （202404BP090087）
Open Fund Project of Forestry Discipline and Key Laboratory of Southwest Forestry University(LXXK-2024M14) （LXXK-2024M14）
