Cloning and Functional Analysis of AeF3H Gene in Okra

doi:10.13560/j.cnki.biotech.bull.1985.2025-0837

Abstract

Abstract:

Objective The AeF3H was cloned from okra (Abelmoschus esculentus), and its biological function was characterized via transformation of Arabidopsis thaliana, aiming to provide a theoretical basis for further elucidating the flavonoid accumulation in okra. Method The primers were designed based on the A. esculentus transcriptome data, and the seed’s cDNA was used as template to amplify the AeF3H gene by PCR. The sequence features were analyzed using bioinformatics. The expression characteristics of AeF3H gene in different tissues of okra were analyzed using real-time fluorescence quantitative PCR (RT-qPCR). A 35S::AeF3H::YFP overexpressing vector was constructed and transformed into Arabidopsis via the floral dip method. Transgenic plants were screened, and the total flavonoids contents in the T₃lines were determined by Al(NO₃)₃ colorimetric method. The subcellular localization of AeF3H was analyzed by transient expression in Arabidopsis protoplasts. Result The open reading frame (ORF) of AeF3H was 1 101 bp in length and encoded a 366-amino acid protein. The genomic DNA spanned 1 304 bp, containing 3 exons and 2 introns. The deduced protein molecular weight was 41.08 kD and its theoretical isoelectric point was 5.32. Phylogenetic tree analysis revealed that AeF3H demonstrated the closest homology to the F3H from Hibiscus trionum. RT-qPCR results showed that AeF3H had a tissue-specific expression profile, with particularly high expressions detected in young seeds and buds, and the lowest levels detected in the stem and pod. Subcellular localization showed that the AeF3H protein was located in both nucleus and cytosol. Arabidopsis plants overexpressing AeF3H presented a 4.75-fold increase in total flavonoid content compared to wild-type plants. Conclusion AeF3H has tissue-specific expression, and its protein is localized to both the nucleus and cytoplasm. AeF3H plays a crucial role in the flavonoid biosynthetic pathway in okra. The overexpression of AeF3H may significantly increase total flavonoid content in transgenic Arabidopsis.

Key words: Abelmoschus esculentus L., AeF3H, flavanone 3-hydroxylase, gene cloning, functional verification, total flavonoids, tissue expression pattern

SUN Ting, ZHANG Yan, LIU Yu-shan, FENG Yuan-yuan, QIN Heng-shan, ZHANG Jun, HE Xiao-gang, ZHANG Jing-rong. Cloning and Functional Analysis of AeF3H Gene in Okra[J]. Biotechnology Bulletin, 2026, 42(4): 153-160.

Figures/Tables 7

Table 1 Primers' sequences used in this study

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	用途 Purpose
F3H-F	CCCAACTAAACATAGAAACACTCAC	ORF序列扩增
F3H-R	ACTAAGACAATGGCACCAAGCAC	Amplification of ORF sequence
F3HYFP-F	CCGCTCGAGATGGCTCCTTCCACTCTCAC （Xoh I）	载体构建
F3HYFP-R	CGCGGATCCTGCAAGGATTTGCTCTAGCG （BamH I）	Vector construction
YFP-F	GCGACGTAAACGGCCACAAGT	转基因拟南芥的PCR鉴定
YFP-R	CAGCTCGTCCATGCCGAGAGT	PCR identification of transgenic Arabidopsis
qeIF4a-F	ATGCATATGGTTTTGAGAAGCC	秋葵内参基因
qeIF4a-R	AAAGTTGCAGTCTTCCCAGTTC	Reference gene in A. esculentus
qF3H-F	AGTTTTTCGCTTTGCCTGCT	实时荧光定量PCR
qF3H-R	TCTCGCTGTATTCCTTTGTCAC	Real-time quantitative PCR
qAtEF1α-F	ATTGACAGGCGTTCTGGTAAG	拟南芥内参基因
qAtEF1α-R	CAGCAACGGTCTGCCTCAT	Reference gene in Arabidopsis

Fig. 1 Amplification electrophoresis map of AeF3H gene

Fig. 2 Sequence alignment (A) and phylogenetic tree analysis (B) of AeF3HIn Fig. A, red boxes indicate the ferrous iron ligation motif HXDX55H and a 2oxoglutarate (2-ODD) binding motif RLS. Black triangles (▲) indicate the conserved amino acid residues in F3Hs. Five conserved motifs in plant 2-oxoglutarate-dependent dioxygenase are also marked

Fig. 3 Expression pattern analysis of the AeF3H geneDifferent lowercase letters indicate significant differences (P<0.05). The same below

Fig. 4 Subcellular localization analysis of AeF3H (Scale = 10 μm)

Fig. 5 Phenotypes (A) and RT-qPCR analysis (B) of wild-type and transgenic Arabidopsis plants

Fig. 6 Effects of overexpressing AeF3H on the total flavonoids contents in ArabidopsisA: Standard curve of rutin. B: Determination of total flavonoids in wild-type and transgenic plants

References 31

[1]	Romdhane MH, Chahdoura H, Barros L, et al. Chemical composition, nutritional value, and biological evaluation of Tunisian okra pods (Abelmoschus esculentus L. moench) [J]. Molecules, 2020, 25(20): 4739.
[2]	Gakhar M, Singh L, Routh S, et al. Evaluation of anti-cancer potential of Abelmoschus esculentus (Okra) [J]. Protein Pept Lett, 2025: 32(8) : 575-583.
[3]	Panighel G, Ferrarese I, Lupo MG, et al. Investigating the in vitro mode of action of okra (Abelmoschus esculentus) as hypocholesterolemic, anti-inflammatory, and antioxidant food [J]. Food Chem Mol Sci, 2022, 5: 100126.
[4]	Saatchi A, Aghamohammadzadeh N, Beheshtirouy S, et al. Anti-hyperglycemic effect of Abelmoschus culentesus (Okra) on patients with diabetes type 2: a randomized clinical trial [J]. Phytother Res, 2022, 36(4): 1644-1651.
[5]	郑晟, 毛玉珊, 张腾国, 等. 柠条锦鸡儿F3H基因克隆及功能分析 [J]. 广西植物, 2017, 37(6): 723-733.
	Zheng S, Mao YS, Zhang TG, et al. Cloning and expression analysis of F3H gene in Caragana korshinskii [J]. Guihaia, 2017, 37(6): 723-733.
[6]	朱三明, 郑敏敏, 田恬, 等. 植物次生代谢途径与调控研究进展 [J]. 植物生理学报, 2023, 59(12): 2188-2216.
	Zhu SM, Zheng MM, Tian T, et al. Research progress on plant secondary metabolism and regulation [J]. Plant Physiol J, 2023, 59(12): 2188-2216.
[7]	Liu WX, Feng Y, Yu SH, et al. The flavonoid biosynthesis network in plants [J]. Int J Mol Sci, 2021, 22(23): 12824.
[8]	段玥彤, 王鹏年, 张春宝, 等. 植物黄烷酮-3-羟化酶基因研究进展 [J]. 生物技术通报, 2022, 38(6): 27-33.
	Duan YT, Wang PN, Zhang CB, et al. Research progress in plant flavanone-3-hydroxylase gene [J]. Biotechnol Bull, 2022, 38(6): 27-33.
[9]	Zhu SM, Cui MY, Zhao Q. Characterization of the 2ODD genes of DOXC subfamily and its members involved in flavonoids biosynthesis in Scutellaria baicalensis [J]. BMC Plant Biol, 2024, 24(1): 804.
[10]	Li XH, Park NI, Kim YB, et al. Accumulation of flavonoids and expression of flavonoid biosynthetic genes in Tartary and rice-Tartary buckwheat [J]. Process Biochem, 2012, 47(12): 2306-2310.
[11]	Sunil L, Shetty NP. Biosynthesis and regulation of anthocyanin pathway genes [J]. Appl Microbiol Biotechnol, 2022, 106(5/6): 1783-1798.
[12]	Dai MJ, Kang XR, Wang YQ, et al. Functional characterization of flavanone 3-hydroxylase (F3H) and its role in anthocyanin and flavonoid biosynthesis in mulberry [J]. Molecules, 2022, 27(10): 3341.
[13]	Xiong S, Tian N, Long JH, et al. Molecular cloning and characterization of a flavanone 3-hydroxylase gene from Artemisia annua L [J]. Plant Physiol Biochem, 2016, 105: 29-36.
[14]	曹鑫娴, 石佳琪, 王霜, 等. 苦荞黄烷酮3-羟化酶基因FtF3H及其启动子的克隆与功能分析 [J]. 四川农业大学学报, 2023, 41(4): 582-591, 639.
	Cao XX, Shi JQ, Wang S, et al. Cloning and functional analysis of flavanone 3-hydroxylase gene FtF3H and its promoter in Fagopyrum tataricum [J]. J Sichuan Agric Univ, 2023, 41(4): 582-591, 639.
[15]	Song XY, Diao JJ, Ji J, et al. Molecular cloning and identification of a flavanone 3-hydroxylase gene from Lycium chinense, and its overexpression enhances drought stress in tobacco [J]. Plant Physiol Biochem, 2016, 98: 89-100.
[16]	赵莹, 杨欣宇, 赵晓丹, 等. 植物类黄酮化合物生物合成调控研究进展 [J]. 食品工业科技, 2021, 42(21): 454-463.
	Zhao Y, Yang XY, Zhao XD, et al. Research progress on regulation of plant flavonoids biosynthesis [J]. Sci Technol Food Ind, 2021, 42(21): 454-463.
[17]	Yang J, Chen XQ, Rao SQ, et al. Identification and quantification of flavonoids in okra (Abelmoschus esculentus L. moench) and antiproliferative activity in vitro of four main components identified [J]. Metabolites, 2022, 12(6): 483.
[18]	Cui YT, Wu JG, Chen YY, et al. Optimization of near-infrared reflectance models in determining flavonoid composition of okra (Abelmoschus esculentus L.) pods [J]. Food Chem, 2023, 418: 135953.
[19]	Guebebia S, Espinosa-Ruiz C, Zourgui L, et al. Effects of okra (Abelmoschus esculentus L.) leaves, fruits and seeds extracts on European sea bass (Dicentrarchus labrax) leukocytes, and their cytotoxic, bactericidal and antioxidant properties [J]. Fish Shellfish Immunol, 2023, 138: 108799.
[20]	Wang RY, Li W, He Q, et al. The genome of okra (Abelmoschus esculentus) provides insights into its genome evolution and high nutrient content [J]. Hortic Res, 2023, 10(8): uhad120.
[21]	Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [J]. Plant J, 1998, 16(6): 735-743.
[22]	Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis [J]. Nat Protoc, 2007, 2(7): 1565-1572.
[23]	Zhao FF, Zhao T, Deng LB, et al. Visualizing the essential role of complete virion assembly machinery in efficient hepatitis C virus cell-to-cell transmission by a viral infection-activated split-intein-mediated reporter system [J]. J Virol, 2017, 91(2): e01720-16.
[24]	杨飞芸, 武燕燕, 崔爽, 等. 异源表达CiRS基因通过生成白藜芦醇增强拟南芥的抗氧化能力 [J]. 中国生物工程杂志, 2017, 37(12): 27-33.
	Yang FY, Wu YY, Cui S, et al. Heterologous expression of CiRS gene enhances the antioxidant capacity of Arabidopsis by increasing the content of resveratrol [J]. China Biotechnol, 2017, 37(12): 27-33.
[25]	Zhang JR, Feng YY, Yang MJ, et al. Systematic screening and validation of reliable reference genes for qRT-PCR analysis in okra (Abelmoschus esculentus L.) [J]. Sci Rep, 2022, 12(1): 12913.
[26]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method [J]. Methods, 2001, 25(4): 402-408.
[27]	李萍, 王若曦, 王欢. 黄酮3-羟化酶(F3H)的生物信息学分析 [J]. 生物学杂志, 2015, 32(6): 25-29.
	Li P, Wang RX, Wang H. Bioinformatics analysis of flavanone-3-hydroxylase (F3H) [J]. J Biol, 2015, 32(6): 25-29.
[28]	张欣宇, 董潞娜, 曹浩, 等. 不同植物源的黄烷酮-3-羟化酶的催化特性 [J]. 微生物学通报, 2022, 49(3): 875-887.
	Zhang XY, Dong LN, Cao H, et al. Catalytic properties of flavanone-3-hydroxylase from different plants [J]. Microbiol China, 2022, 49(3): 875-887.
[29]	Li CC, Liu SH, Yao XH, et al. PnF3H, a flavanone 3-hydroxylase from the Antarctic moss Pohlia nutans, confers tolerance to salt stress and ABA treatment in transgenic Arabidopsis [J]. Plant Growth Regul, 2017, 83(3): 489-500.
[30]	Mao YY, Luo JJ, Cai ZP. Biosynthesis and regulatory mechanisms of plant flavonoids: a review [J]. Plants, 2025, 14(12): 1847.
[31]	Quattrocchio F, Wing JF, Va K, et al. Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes [J]. Plant J, 1998, 13(4): 475-488.