比利时杜鹃花类黄酮3'-羟化酶（F3'H）基因克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0034

摘要/Abstract

摘要：

【目的】 类黄酮3'-羟化酶（flavanone 3'-hydroxylase，F3'H）是植物花青素合成过程中的关键酶，探究比利时杜鹃花（Rhododendron hybridum Hort）F3'H基因的功能及表达特性。【方法】 以比利时杜鹃花不同发育时期的花瓣及盛花期的根、茎、叶为实验材料，从比利时杜鹃花转录本数据库中筛选类黄酮生物合成通路关键酶类黄酮3'-羟化酶基因序列信息并进行生物信息学分析，利用反转录（RT-PCR）技术克隆RhF3'H基因；利用紫外分光光度计测定了比利时杜鹃花花瓣不同时期的花青素含量，利用RT-qPCR技术对不同发育时期花瓣和成熟期的不同组织进行RhF3'H基因表达量分析；采用Gateway技术构建过表达载体35S:RhF3'H-GFP重组载体进行亚细胞定位验证；构建p1302-RhF3'H过表达载体对比利时杜鹃花花瓣进行侵染。【结果】 成功获得比利时杜鹃花RhF3'H基因长度为1 557 bp,编码518个氨基酸，具有保守的F3'H结构域，属于P450超家族；系统进化树分析显示，比利时杜鹃花RhF3'H与龙眼和荔枝F3'H蛋白亲缘关系最近；RT-qPCR结果显示，RhF3'H在比利时杜鹃花不同花瓣时期和根、茎、叶组织中均有表达，在不同花瓣发育时期中，盛开期和衰败期中RhF3'H基因的表达量较高，与花青素含量结果相一致；亚细胞定位分析显示，RhF3'H主要存在于细胞膜上；成功构建P1302-RhF3'H瞬时过表达载体，相较于CK和p1302，p1302-RhF3'H在杜鹃花花瓣中显著高表达，其花青素含量也显著增加。【结论】 RhF3'H基因在花瓣细胞膜中表达，表达模式与花青素积累趋势一致；过表达RhF3'H基因促进了花青素的合成。

关键词: 比利时杜鹃花, 类黄酮3'-羟化酶（F3'H）, 花青素, 亚细胞定位, 瞬时表达

Abstract:

【Objective】 The flavonoid 3'-hydroxylase（F3'H）is a key enzyme in the synthesis of anthocyanins in plants, and the function and expression characteristics of F3'H gene in Rhododendron hybridum Hort were investigated.【Method】 The petals and roots, stems and leaves of R. hybridum Hort at different developmental stages were used as experimental materials. The flavonoid 3'-hydroxylase gene sequence information of flavonoid biosynthesis pathway was screened from the R. hybridum Hort transcript database and bioinformatics analysis was performed, and the RhF3'H gene was cloned by reverse transcription（RT-PCR）technology. The anthocyanin content in the petals of R. hybridum Hort at different stages was determined by ultraviolet spectrophotometer, and the expression of RhF3'H gene was analyzed by RT-qPCR technology on different tissues of petals at different development stages and maturity stages. The overexpression vector 35S:RhF3'H-GFP recombinant vector was constructed by Gateway technology for subcellular localization verification, and p1302-RhF3'H overexpression vector was constructed to infect the petals of R. hybridum Hort.【Result】 The RhF3'H gene was 1 557 bp in length and encoded 518 amino acids, and the protein encoded by RhF3'H belonged to the P450 superfamily with a conserved F3'H domain. Phylogenetic tree analysis showed that RhF3'H was the most closely related to Dimocarpus longan and Litchi chinensis F3'H proteins. The results of RT-qPCR showed that RhF3'H was expressed in different petal stages and roots, stems, and leaf tissues of R. hybridum Hort. The expressions of RhF3'H gene were higher in the blooming and decaying stages of different petal development stages, which was consistent with the anthocyanin content, and subcellular localization analysis showed that RhF3'H mainly existed on the cell membrane. Compared with CK and p1302, p1302-RhF3'H was significantly higher in the petals of R. hybridum Hort and its anthocyanin content significantly increased.【Conclusion】 The RhF3'H gene was expressed in the petal cell membrane, and the expression pattern was consistent with the accumulation trend of anthocyanins. Overexpression of the RhF3'H gene promotes the synthesis of anthocyanins.

Key words: Rhododendron hybridum Hort, flavanone 3'-hydroxylase, anthocyanidin, subcellular localization, instantaneous expression.

吴泽航, 杨中义, 鄢毅铖, 贾永红, 吴月燕, 谢晓鸿. 比利时杜鹃花类黄酮3'-羟化酶（F3'H）基因克隆及功能分析[J]. 生物技术通报, 2024, 40(6): 251-259.

WU Ze-hang, YANG Zhong-yi, YAN Yi-cheng, JIA Yong-hong, WU Yue-yan, XIE Xiao-hong. Cloning and Functional Analysis of Flavonoid 3'-hydroxylase（F3'H）Gene in Rhododendron hybridum Hort[J]. Biotechnology Bulletin, 2024, 40(6): 251-259.

图/表 6

图1 比利时杜鹃花花瓣不同花期（A）和成熟期组织（B）

Fig. 1 Petal tissues of different flowering（A）and mature（B）stages of R. hybridum Hort.

表1 RhF3'H基因克隆及表达分析引物

Table 1 Primers for RhF3'H gene cloning and expression analysis

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
F3'H-F1	ATGACTTCTTCTTTGGCTCTTGTTC
F3'H-R1	TCAGCCCCGATACAGATGGG
F3'H-F2	GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGACTTCTTCTTTGGCTCTTGTTC
F3'H-R2	GGGGACCACTTTGTACAAGAAAGCTGGGTCGCCCCGATACAGATGGGC
F3'H-F3	AGTCCTAGCCAAAGCCCAAC
F3'H-R3	GAGAGAGAGCGGTGTCGATG
Actin-F	CACTGGTGTCATGGTTGGGA
Actin-R	CTCTTCAGGAGCAACACGGA
F3'H-F4	ACGGGGGACTCTTGACCATGGATGACTTCTTCTTTGGCTCTTGTTC
F3'H-R4	TACTAGTCAGATCTACCATGGGCCCCGATACAGATGGGC

图2 比利时杜鹃花与其他物种F3'H蛋白的比对和系统进化树分析 A: RhF3'H蛋白的保守域预测；B: F3'H蛋白序列比对（a: F3'H蛋白膜锚定位点；b：底物选择和结合位点；c：血红素结合区域）；C：F3'H蛋白系统进化树分析；其他物种的拉丁名和蛋白：菊花（Chrysanthemum morifolium，AB523844.1，CmF3'H）、紫锥菊（Echinacea purpurea，OQ992545.1，EpF3'H）、芍药（Paeonia lactiflora，JQ070803.1，PlF3'H）、石竹（Dianthus caryophyllus，AB778537.1，DcF3'H）、荔枝（Litchi chinensis，QRV61378.1，LcF3'H）、水稻（Oryza sativa，WGJ78734.1，OsF3'H）、龙眼（Dimocarpus longan，QRM13299.1，DlF3'H）、水仙（Narcissus tazetta，AFP95893.1，NtF3'H）、乌桕（Triadica sebifera，QOU08777.1，TsF3'H）、花生（Arachis hypogaea，AEX07282.1，AhF3'H）、三花龙胆（Gentiana triflora，UTD45114.1，GtF3'H）、大豆（Glycine max，NP_001237015.1，GmF3'H）、紫叶李（Prunus cerasifera，AKV89245.1，PcF3'H）

Fig. 2 Alignment and phylogenetic tree analysis of F3'H protein in R. hybridum Hort and other species A: Conserved domain prediction of RhF3'H protein. B: F3'H protein sequence alignment;（a: F3'H protein membrane anchor site; b: substrate selection and binding sites; c: Heme-binding region.）.C: Phylogenetic tree analysis of F3'H protein. Latin names and genes of other species:（Chrysanthemum morifolium，AB523844.1，CmF3'H），（Echinacea purpurea，OQ992545.1，EpF3'H），（Paeonia lactiflora，JQ070803.1，PlF3'H），（Dianthus caryophyllus，AB778537.1，DcF3'H）,（Litchi chinensis，QRV61378.1，LcF3'H）,（Oryza sativa，WGJ78734.1，OsF3'H）,（Dimocarpus longan，QRM13299.1，DlF3'H）,（Narcissus tazetta，AFP95893.1，NtF3'H）,（Triadica sebifera，QOU08777.1，TsF3'H）,（Arachis hypogaea，AEX07282.1，AhF3'H）,（Gentiana triflora，UTD45114.1，GtF3'H）,（Glycine max，NP_001237015.1，GmF3'H）,（Prunus cerasifera，AKV89245.1，PcF3'H）

图3 比利时杜鹃花总花青素含量与F3'H相对表达量分析 A：花瓣不同时期总花青素含量；B：花期和不同组织的F3'H相对表达量。S1：花苞期；S2：初开期；S3：盛开期；S4：衰败期。不同小写字母表示差异显著（P<0.05），下同

Fig. 3 Relative expressions of total anthocyanin content and F3'H in R. hybridum Hort. A：Total anthocyanin content of petals at different periods. B：Relative expression of F3'H at flowering stages and in different tissues. S1: Flower bud stage; S2: initial opening; S3: in full bloom; S4: decay period. Different lowercase letters indicate significant difference (P<0.05). The same below

图4 RhF3'H亚细胞定位

Fig. 4 Subcellular localization for RhF3'H

图5 RhF3'H基因瞬时过表达的总花青素和相对表达量 A：瞬时过表达花瓣表型图；B：瞬时过表达总花青素含量变化图；C：瞬时过表达RhF3'H相对表达量变化图；CK:未处理；p1302:空载；p1302-RhF3'H：目的基因

Fig. 5 Transiently overexpressed total anthocyanins and relative expressions of RhF3'H gene A: Phenotypic diagram of petal via the transient overexpression. B: Diagram of total anthocyanin content changing via transient overexpression. C: Relative expression of RhF3'H via transient overexpression. CK: Not processed. p1302: Idle; p1302-RhF3'H: Gene of interest

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