黄秋葵AeF3H基因的克隆与功能分析

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

摘要/Abstract

摘要：

目的从黄秋葵（Abelmoschus esculentus L.）中克隆黄烷酮3-羟化酶基因（AeF3H），通过拟南芥遗传转化鉴定其生物学功能，为阐明黄秋葵类黄酮累积的调控机理提供理论基础。方法根据黄秋葵转录组数据设计引物，以秋葵籽cDNA为模板，通过PCR扩增获得AeF3H基因；通过生物信息学分析AeF3H的基本特征；采用实时荧光定量PCR（RT-qPCR）分析其组织表达特性；构建35::AeF3H::YFP过表达载体，通过花序浸染法转化拟南芥，采用硝酸铝比色法测定T₃代拟南芥转基因株系中总黄酮含量变化；利用拟南芥原生质体瞬时表达技术分析AeF3H亚细胞定位。结果 AeF3H的ORF长1 101 bp，编码366个氨基酸；基因组序列长1 304 bp，含3个外显子和2个内含子。推测其蛋白的相对分子量为41.08 kD，理论等电点为5.32。系统进化树分析发现，AeF3H与木槿（Hibiscus trionum）的F3H亲缘关系最近。RT-qPCR结果表明，AeF3H具有一定的组织表达特异性，在嫩籽和花蕾中的表达量最高，在茎和果荚中的表达量最低。亚细胞定位显示其蛋白同时定位于细胞核和细胞质中。过表达AeF3H的拟南芥植株，其总黄酮含量为野生型的4.75倍。结论 AeF3H具有一定的组织表达特异性，其蛋白定位于细胞核和细胞质中。AeF3H在黄秋葵类黄酮生物合成代谢途径中发挥着重要作用，过量表达AeF3H能够显著增加转基因拟南芥的总黄酮含量。

关键词: 黄秋葵, AeF3H, 黄烷酮3-羟化酶, 基因克隆, 功能验证, 总黄酮, 组织表达模式

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

孙婷, 张艳, 刘玉珊, 冯媛媛, 秦恒山, 张军, 何小岗, 张景荣. 黄秋葵AeF3H基因的克隆与功能分析[J]. 生物技术通报, 2026, 42(4): 153-160.

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.

图/表 7

表 1 本实验所用的引物序列

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

图1 AeF3H基因扩增电泳图1： PCR product. M： DL2000 marker

Fig. 1 Amplification electrophoresis map of AeF3H gene

图2 AeF3H的序列比对（A）与系统进化树分析（B）图A中，红色方框标示出亚铁离子配位基序HXDX55H及2-氧代戊二酸（2-ODD）结合基序RLS，保守氨基酸残基用黑色三角形（▲）标记；同时标记出植物2-氧戊二酸依赖型双加氧酶中的5个保守基序

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

图3 AeF3H基因的表达模式分析不同字母表示差异显著（P<0.05）。下同

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

图4 AeF3H的亚细胞定位分析

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

图5 野生型和转基因拟南芥植株表型（A）和RT-qPCR分析（B）

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

图6 过量表达AeF3H对拟南芥总黄酮含量的影响A：芦丁标准曲线；B：野生型植株和转基因植株总黄酮含量的测定

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

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