生物技术通报2026，Vol.42Issue(4)：153-160,8.DOI:10.13560/j.cnki.biotech.bull.1985.2025-0837
黄秋葵AeF3H基因的克隆与功能分析
Cloning and Functional Analysis of AeF3H Gene in Okra
摘要
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 T3 lines 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.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Key words
Abelmoschus esculentus L./AeF3H/flavanone 3-hydroxylase/gene cloning/functional verification/total flavonoids/tissue expression pattern引用本文复制引用
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):153-160,8.基金项目
四川省农业科学院"5+1"农业前沿技术攻关项目(5+1QYGG005),四川省省级公益性科研单位基本科研业务费专项资金项目(24JBKY07) （5+1QYGG005）
