Cloning and Functional Analysis of Flavonoid 3'-hydroxylase（F3'H）Gene in Rhododendron hybridum Hort

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

Abstract

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.

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.

Figures/Tables 6

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

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

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）

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

Fig. 4 Subcellular localization for RhF3'H

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

References 32

[1]	Li ZL, Yang QF, Dong X, et al. Transcriptome analysis of flower color variation in five Rhododendron species(Ericaceae)[J]. Braz J Bot, 2021, 44(3): 685-695.
[2]	Sun W, Zhou NN, Wang YH, et al. Characterization and functional analysis of RdDFR1 regulation on flower color formation in Rhododendron delavayi[J]. Plant Physiol Biochem, 2021, 169: 203-210.
[3]	Sakuta M, Tanaka A, Iwase K, et al. Anthocyanin synthesis potential in betalain-producing Caryophyllales plants[J]. J Plant Res, 2021, 134(6): 1335-1349. doi: 10.1007/s10265-021-01341-0 pmid: 34477986
[4]	Fritsch H, Grisebach H. Biosynthesis of cyanidin in cell cultures of Haplopappus gracilis[J]. Phytochemistry, 1975, 14(11): 2437-2442.
[5]	Holton TA, Brugliera F, Lester DR, et al. Cloning and expression of cytochrome P450 genes controlling flower colour[J]. Nature, 1993, 366(6452): 276-279.
[6]	Schoenbohm C, Martens S, Eder C, et al. Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450 enzyme[J]. Biol Chem, 2000, 381(8): 749-753. pmid: 11030432
[7]	赵志常, 高爱平, 黄建峰, 等. 芒果(Mangifera indica)F3'H基因的克隆及其表达分析[J]. 西南农业学报, 2016, 29(4):903-908.
	Zhao ZC, Gao AP, Huang JF, et al. Cloning and expression analysis of F3'H gene from mango(Mangifera indica)[J]. Southwest China Journal of Agricultural Sciences, 2016, 29(4): 903-908.
[8]	景宗慧, 尹梦娇, 王前, 等. 芡叶类黄酮-3'-羟化酶(F3'H)基因表达分析及功能验证[J]. 中国中药杂志, 2021, 46(18): 4712-4720.
	Jing ZH, Yin MJ, Wang Q, et al. Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox[J]. China J Chin Mater Med, 2021, 46(18): 4712-4720.
[9]	黄文坤, 程红梅, 郭建英, 等. 紫茎泽兰类黄酮3'-羟化酶基因的克隆、序列分析和原核表达[J]. 植物生理学通讯, 2007, 43(5): 821-826.
	Huang WK, Cheng HM, Guo JY, et al. Cloning, sequence analysis and expression in Escherichia coli of flavonoid 3'-hydroxylase gene of Eupatorium adenophorum sprengel[J]. Plant Physiol Commun, 2007, 43(5): 821-826.
[10]	范月婷, 辛士超, NAYCHI Koko, 等. 橡胶树F3'H基因克隆及其功能分析[J]. 热带作物学报, 2020, 41(9): 1733-1740. doi: 10.3969/j.issn.1000-2561.2020.09.002
	Fan YT, Xin SC, Naychi K, et al. Cloning and functional verification of rubber tree F3'H gene[J]. Chin J Trop Crops, 2020, 41(9): 1733-1740.
[11]	Yu HF, Wang JS, Shen YS, et al. A 43 bp-deletion in the F3'H gene reducing anthocyanins is responsible for keeping buds green at low temperatures in broccoli[J]. Int J Mol Sci, 2023, 24(14): 11391.
[12]	Nitarska D, Boehm R, Debener T, et al. First genome edited poinsettias: targeted mutagenesis of flavonoid 3'-hydroxylase using CRISPR/Cas9 results in a colour shift[J]. Plant Cell Tissue Organ Cult, 2021, 147(1): 49-60.
[13]	Han YP, Vimolmangkang S, Soria-Guerra RE, et al. Ectopic expression of apple F3'H genes contributes to anthocyanin accumulation in the Arabidopsis tt7 mutant grown under nitrogen stress[J]. Plant Physiol, 2010, 153(2): 806-820.
[14]	Mehrtens F, Kranz H, Bednarek P, et al. The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis[J]. Plant Physiol, 2005, 138(2): 1083-1096. doi: 10.1104/pp.104.058032 pmid: 15923334
[15]	刘小飞, 于波, 黄丽丽, 等. 杜鹃红山茶实时定量PCR内参基因筛选及验证[J]. 广东农业科学, 2020, 47(12): 203-211.
	Liu XF, Yu B, Huang LL, et al. Screening and validation of reference genes of Camellia azalea by quantitative real-time PCR[J]. Guangdong Agric Sci, 2020, 47(12): 203-211.
[16]	侯瑞泽, 鲍悦, 陈启亮, 等. 普通白菜PRR5的克隆、表达及功能验证[J]. 生物技术通报, 2023, 39(10): 128-135. doi: 10.13560/j.cnki.biotech.bull.1985.2023-0338
	Hou RZ, Bao Y, Chen QL, et al. Cloning, expression and functional identification of PRR5 gene in pakchoi[J]. Biotechnol Bull, 2023, 39(10): 128-135.
[17]	刘海峰, 杨成君, 赵权, 等. 山葡萄中类黄酮3'-羟化酶基因(F3'H)cDNA的克隆和分析[J]. 植物生理学通讯, 2009, 45(12): 1186-1190.
	Liu HF, Yang CJ, Zhao Q, et al. cDNA cloning and analysis of flavonoid 3'-hydroxylase(F3'H)in vitis amurensis Rupr.[J]. Plant Physiology Journal. 2009, 45(12): 1186-1190.
[18]	陈盼, 曹天骏, 戴好富, 等. 海南龙血树类黄酮3'-羟化酶基因(DcF3'H)的克隆与表达分析[J]. 热带作物学报, 2016, 37(3): 568-575.
	Chen P, Cao TJ, Dai HF, et al. Cloning and expression analysis of flavonoid 3'-hydroxylase(F3'H)gene in Dracaena cambodiana Pierre ex gagnep[J]. Chin J Trop Crops, 2016, 37(3): 568-575.
[19]	康美玲, 冯凯, 段希, 等. 水芹类黄酮3'-羟化酶基因的克隆与表达特性分析[J]. 植物生理学报, 2018, 54(2): 282-290.
	Kang ML, Feng K, Duan X, et al. Cloning and expression analysis of flavonoid 3'-hydroxylase in Oenanthe javanica[J]. Plant Physiol J, 2018, 54(2): 282-290.
[20]	何海旺, 潘华清, 张铙丹, 等. 飞机草类黄酮3'-羟化酶基因(CoF3'H)的克隆及其在烟草中的表达[J]. 作物学报, 2015, 41(3):479-486.
	He HW, Pan HQ, Zhang ND, et al. Chromolaena odorata flavonoid 3'-hydroxylase gene cloning and its expression in tobacco[J]. Acta Agronomica Sinica, 2015, 41(3): 479-486.
[21]	李永华, 蒲天珍, 周佩娜, 等. 大叶蛇葡萄类黄酮-3'-羟化酶基因(F3'H)的克隆及生物信息学分析[J]. 分子植物育种, 2021, 19(18): 5984-5993.
	Li YH, Pu TZ, Zhou PN, et al. Cloning and bioinformatics analysis of flavonoid-3'-hydroxylase(F3'H)gene in Ampelopsis megalophylla[J]. Mol Plant Breed, 2021, 19(18): 5984-5993.
[22]	Yamazaki S, Sato K, Suhara K, et al. Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s[J]. J Biochem, 1993, 114(5): 652-657. pmid: 8113216
[23]	Murakami K, Mihara K, Omura T. The transmembrane region of microsomal cytochrome P450 identified as the endoplasmic reticulum retention signal[J]. J Biochem, 1994, 116(1): 164-175. pmid: 7798174
[24]	Boddu J, Svabek C, Sekhon R, et al. Expression of a putative flavonoid 3'-hydroxylase in sorghum mesocotyls synthesizing 3-deoxyanthocyanidin phytoalexins[J]. Physiol Mol Plant Pathol, 2004, 65(2): 101-113.
[25]	蒋宝鑫, 杨国霞, 吕思佳, 等. 比利时杜鹃花花青素合成酶基因(RhANS)克隆及其功能分析[J]. 核农学报, 2023, 37(3): 449-460. doi: 10.11869/j.issn.1000-8551.2023.03.0449
	Jiang BX, Yang GX, Lyu SJ, et al. Cloning and functional analysis of anthocyanin synthase gene(RhANS)in Rhododendron hybridum hort[J]. J Nucl Agric Sci, 2023, 37(3): 449-460.
[26]	吴红松, 秋田祐介. 松果菊F3'H基因的克隆及表达分析[J]. 生物化工, 2022, 8(3): 84-86.
	Wu HS, Yusuke A. Molecular cloning and expression analysis of a flavonoid 3'-hydroxylase gene from Echinacea purpurea[J]. Biol Chem Eng, 2022, 8(3): 84-86.
[27]	王文丽, 吴致君, 刘志薇, 等. 茶树类黄酮3'-羟化酶基因的克隆与表达特性分析[J]. 茶叶科学, 2017, 37(1): 108-118.
	Wang WL, Wu ZJ, Liu ZW, et al. Cloning and expression analysis of the gene encoding flavonoid 3'-hydroxylase in tea plant(Camellia sinensis)[J]. J Tea Sci, 2017, 37(1): 108-118.
[28]	黄小芳. 基于转录组测序的红盖鳞毛蕨类黄酮3'-羟化酶(F3'H)基因的克隆与表达分析[D]. 上海: 上海师范大学, 2016.
	Huang XF.Flavonoid 3'- hydroxylase(F3'H) gene cloning and expression analysis based on the transcriptome sequencing of Dryopteris erythrosora[D]. Shanghai: Shanghai Normal University, 2016.
[29]	李琳玲, 程华, 陈小玲, 等. 银杏类黄酮3'羟化酶基因的克隆与表达分析[J]. 园艺学报, 2015, 42(4): 643-654.
	Li LL, Cheng H, Chen XL, et al. Molecular cloning, characterization and expression of flavonoid 3' hydroxylase-like protein genefrom Ginkgo biloba[J]. Acta Horticulturae Sinica, 2015, 42(4): 643-654.
[30]	Wu YQ, Wang TL, Xin Y, et al. Overexpression of the GbF3'H1 gene enhanced the epigallocatechin, gallocatechin, and catechin contents in transgenic Populus[J]. J Agric Food Chem, 2020, 68(4): 998-1006.
[31]	Li CL, Yang K, Yang JJ, et al. Tartary buckwheat FtF3'H1 as a metabolic branch switch to increase anthocyanin content in transgenic plant[J]. Front Plant Sci, 2022, 13: 959698.
[32]	李双江, 朱冬寅, 秦东, 等. 苦荞类黄酮3'-羟化酶基因的克隆及其冷胁迫下的组织表达[J]. 中草药, 2014, 45(9): 1300-1306.
	Li SJ, Zhu DY, Qin D, et al. Cloning of flavonoid 3'-hydroxylase gene from Fagopyrum tataricum and its tissue-specific expression under cold stress[J]. Chin Tradit Herb Drugs, 2014, 45(9): 1300-1306.