Cloning, expression analysis and enzyme activity verification of dihydroflavonol 4-reductase from Cistanche tubulosa (Schenk) Wight flower
10.16438/j.0513-4870.2022-1008
- VernacularTitle:管花肉苁蓉花中二氢黄酮醇4-还原酶的克隆、表达分析和酶活性鉴定
- Author:
Hai-ling QIU
1
,
2
;
Fang-ming WANG
3
;
Bo-wen GAO
4
;
Xin-yu MI
1
,
2
;
Ze-kun ZHANG
1
,
2
;
Yu DU
1
,
2
;
She-po SHI
1
,
2
;
Peng-fei TU
3
;
Xiao-hui WANG
1
,
2
Author Information
1. School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 102488, China
2. Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China
3. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Science, Peking University, Beijing 100191, China
4. Baotou Medical College, Baotou 014060, China
- Publication Type:Research Article
- Keywords:
italic>Cistanche tubulosa (Schenk) Wight;
flower color;
ihydroflavonol 4-reductase;
expression analysis;
enzyme activity analysis;
subcellular localization
- From:
Acta Pharmaceutica Sinica
2023;57(4):1079-1089
- CountryChina
- Language:Chinese
-
Abstract:
Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.
