桂花OfbHLH105参与调控类胡萝卜素代谢的研究

doi:10.16420/j.issn.0513-353x.2025-0380

摘要/Abstract

摘要：

花色是桂花重要的观赏性状之一，花瓣类胡萝卜素的组成和含量直接影响桂花花色，其积累受到转录因子的调控。本研究中，从桂花品种‘金球桂’中克隆转录因子OfbHLH105，多序列比对和系统进化树分析结果显示，OfbHLH105具有典型的bHLH保守结构域，且与拟南芥中AtbHLH105亲缘关系最近。亚细胞定位结果显示，OfbHLH105为核定位转录因子。对OfbHLH105在‘金球桂’开花过程中的表达模式进行分析，结果显示其在顶壳期表达量最高。对异源过表达OfbHLH105烟草株系的分析发现，其叶片中类胡萝卜素含量上升，同时类胡萝卜素合成通路基因NtLCYB的表达上调，多个类胡萝卜素合成途径下游基因和降解基因的表达下调。酵母双杂交结果显示，OfbHLH105能形成同源二聚体。基于以上结果推测，OfbHLH105可能通过同源二聚体的形式参与类胡萝卜素的代谢调控。

关键词: 桂花, 类胡萝卜素, 转录因子, bHLH105, 花色

Abstract:

Flower color is one of the important ornamental traits of Osmanthus fragrans. The composition and content of carotenoids in petals directly affect flower color，and their accumulation is regulated by transcription factors. In this study，the transcription factor OfbHLH105 was cloned from the O. fragrans cultivar‘Jinqiugui’. Multiple sequence alignment and phylogenetic analysis showed that OfbHLH105 contains a typical bHLH conserved domain and is most closely related to AtbHLH105 in Arabidopsis thaliana. Subcellular localization analysis indicated that OfbHLH105 is a nucleus-localized transcription factor. Expression analysis during the flowering process revealed that OfbHLH105 has the highest expression at the bud cracking stage. In transgenic tobacco plants over-expressing OfbHLH105，the carotenoid content in leaves increased，accompanied by up-regulation of carotenoid biosynthesis genes NtLCYB，and down-regulation of several downstream biosynthesis and degradation genes. Yeast two-hybrid results showed that OfbHLH105 can form homodimers. These findings suggest that OfbHLH105 may be involved in regulation of carotenoid metabolism through homodimer formation.

Key words: Osmanthus fragrans, carotenoids, transcription factor, bHLH105, flower color

王忆南, 彭琳, 王艺光, 肖政, 邓金萍, 赵宏波. 桂花OfbHLH105参与调控类胡萝卜素代谢的研究[J]. 园艺学报, 2026, 53(8): 2333-2344.

WANG Yi’nan, PENG Lin, WANG Yiguang, XIAO Zheng, DENG Jinping, ZHAO Hongbo. Investigation of OfbHLH105 in Regulation of Carotenoid Metabolism in Osmanthus fragrans[J]. Acta Horticulturae Sinica, 2026, 53(8): 2333-2344.

图/表 9

图1 桂花品种‘金球桂’花朵开放的不同阶段

Fig. 1 Different flowering stages of flower in Osmanthus fragrans‘Jinqiugui’

表1 本研究中所用引物序列

Table 1 Primers used in this study

引物名称 Primer name	引物序列（5′-3′） Primer sequences	目的 Purpose
OfbHLH105-F	ATGGTTTCCCCGGAAAAAACG	基因克隆 Gene cloning
OfbHLH105-R	TTATGCAACTGGTGGGCGAA	基因克隆 Gene cloning
35S::OfbHLH105-F	CCGcatatgGTTTCCCCGGAAAAAAC	植物过表达载体构建 Construction of plant overexpression vectors
35S::OfbHLH105-R	TACCggatccTTATGCAACTGGTGGGCGAAGCA
pORE-R4-F	GACGCACAATCCCACTATCC
pORE-R4-R	TTGGGACAACTCCAGTGAAA
qRT-OfbHLH105-F	ATGTCGCTGTTCCTGAAGGG	实时荧光定量PCR（qRT-PCR） Real-time quantitative PCR
qRT-OfbHLH105-R	TTCAAGGGTTGCACAGACCA
qRT-OfACT-F	CCCAAGGCAAACAGAGAAAAAAT
qRT-OfACT-R	ACCCCATCACCAGAATCAAGAA
AD-OfbHLH105-F	GGCcatatgGTTTCCCCGGAAAAAACGAATTG	酵母双杂交 Yeast two-hybrid assay
AD-OfbHLH105-R	CGGggatccTTATGCAACTGGTGGGCGAAGCA
BD-OfbHLH105-F	CCcatatgATGGTTTCCCCGGAAAAAAC
BD-OfbHLH105-R	CGggatccCTTATGCAACTGGTGGGCGAAG

图2 桂花OfbHLH105克隆及生物信息分析 A：基因克隆电泳图；B：OfbHLH105与同源蛋白的氨基酸多序列比对图；C：OfbHLH105蛋白二级结构；D：OfbHLH105蛋白亲疏水性。Of：桂花；Oe：油橄榄；Nt：烟草；At：拟南芥

Fig. 2 Cloning and bioinformatics analysis of the OfbHLH105 gene A：Gel electrophoresis of gene cloning；B：Multiple sequence alignment of OfbHLH105 amino acid sequences；C：Secondary structure prediction of the OfbHLH105 protein；D：Hydrophobicity analysis of the OfbHLH105 protein. Of：Osmanthus fragrans；Oe：Olea europaea；Nt：Nicotiana tabacum；At：Arabidopsis thaliana

图3 桂花OfbHLH105与拟南芥bHLH家族蛋白的系统进化分析

Fig. 3 Phylogenetic analysis of OfbHLH105 and Arabidopsis thaliana bHLH proteins

图4 OfbHLH105在‘金球桂’花开放过程中的表达量 S1：圆珠期；S2：顶壳期；S3：铃梗期；S4：初花期；S5：盛花期；S6：盛花末期。不同小写字母表示开花时期间差异显著（P < 0.05）

Fig. 4 Expression levels of OfbHLH105 during the flowering process of‘Jinqiugui’ S1：Round bead stage；S2：Bud cracking stage；S3：Linggeng stage；S4：Initial flowering stage；S5：Full flowering stage；S6：Late flowering stage. Different lowercase letters indicate significant differences between flowering stages at the P < 0.05 level

图5 桂花OfbHLH105蛋白在本氏烟草中的亚细胞定位 35S::GFP为对照

Fig. 5 Subcellular localization of OfbHLH105 protein in Nicotiana benthamiana 35S::GFP is the control

图6 OfbHLH105过表达烟草的叶片表型与类胡萝卜素含量分析 A：烟草表型；B：半定量RT-PCR检测；C：总类胡萝卜素含量测定。** P < 0.01

Fig. 6 Phenotypic analysis and carotenoid content in leaves of tobacco overexpressing OfbHLH105 A：Phenotypic analysis of tobacco；B：RT-PCR analysis；C：Determination of total carotenoid content. ** P < 0.01

图7 OfbHLH105过表达烟草株系类胡萝卜素代谢基因的相对表达量

Fig. 7 Relative expression of genes in carotenoid metabolic pathway in OfbHLH105 overexpressed tobacco strains *P < 0.05，**P < 0.01.

图8 桂花OfbHLH105蛋白酵母双杂交验证阳性对照：ADT7 + BD53；阴性对照：ADT7 + Bdlam

Fig. 8 Yeast two-hybrid assay between OfbHLH105 proteins Positive control：ADT7 + BD53；Negative control：ADT7 + BDlam

参考文献 33

[1]	Chen Jian, Lü Changping, Chen Chentian, Fu Shuangshuang, Chen Haixia. 2009. Components of flower pigments in the petals of different color Gerbera jamesonii. Journal of Hunan Agricultural University(Natural Science), 35 (S1):73-76. (in Chinese)
	陈建, 吕长平, 陈晨甜, 浮双双, 陈海霞. 2009. 不同花色非洲菊品种花色素成分初步分析. 湖南农业大学学报(自然科学版), 35 (S1):73-76.
[2]	Gao F, Robe K, Bettembourg M, Navarro N, Rofidal V, Santoni V, Gaymard F, Vignols F, Roschzttardtz H, Izquierdo E, Dubos C. 2020. The transcription factor bHLH121 interacts with bHLH 105(ILR3)and its closest homologs to regulate iron homeostasis in Arabidopsis. The Plant Cell, 32:508-524. doi: 10.1105/tpc.19.00541 URL
[3]	Han Y, Lu M, Yue S, Li K, Dong M, Liu L, Wang H, Shang F. 2022. Comparative methylomics and chromatin accessibility analysis in Osmanthus fragrans uncovers regulation of genic transcription and mechanisms of key floral scent production. Horticulture Research,9:uhac096.
[4]	Han Y, Wu M, Cao L, Yuan W, Dong M, Wang X, Chen W, Shang F. 2016. Characterization of OfWRKY3,a transcription factor that positively regulates the carotenoid cleavage dioxygenase gene OfCCD4 in Osmanthus fragrans. Plant Molecular Biology, 91:485-496. doi: 10.1007/s11103-016-0483-6 URL
[5]	Hao Y, Oh E, Choi G, Liang Z, Wang Z Y. 2012. Interactions between HLH and bHLH factors modulate light-regulated plant development. Molecular Plant, 5:688-697. doi: 10.1093/mp/sss011 pmid: 22331621
[6]	Heim M A, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey P C. 2003. The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity. Molecular Biology and Evolution, 20:735-747. doi: 10.1093/molbev/msg088 URL
[7]	Hermanns A S, Zhou X, Xu Q, Tadmor Y, Li L. 2020. Carotenoid pigment accumulation in horticultural plants. Horticultural Plant Journal, 6:343-360. doi: 10.1016/j.hpj.2020.10.002
[8]	Ko M R, Song M H, Kim J K, Baek S A, You M K, Lim S H, Ha S H. 2018. RNAi-mediated suppression of three carotenoid-cleavage dioxygenase genes,OsCCD1,4a,and 4b,increases carotenoid content in rice. Journal of Experimental Botany, 69:5105-5116. doi: 10.1093/jxb/ery300 URL
[9]	Li Xiang, Zhong Shiwei, Wang Yiguang, Deng Jinping, Fang Qiu, Zhao Hongbo. 2024. Functional analysis of OfbHLH89 gene in regulating flower opening in Osmanthus fragrans. Acta Horticulturae Sinica, 51 (6):1311-1320. (in Chinese)
	李湘, 钟诗蔚, 王艺光, 邓金萍, 方遒, 赵宏波. 2024. 桂花OfbHLH89参与调控花开放的功能解析. 园艺学报, 51 (6):1311-1320.
[10]	Li Y, Li L, Ding W, Li H, Shi T, Yang X, Wang L, Yue Y. 2020. Genome-wide identification of Osmanthus fragrans bHLH transcription factors and their expression analysis in response to abiotic stress. Environmental and Experimental Botany, 172:103990. doi: 10.1016/j.envexpbot.2020.103990 URL
[11]	Lim S H, Kim D H, Jung J A, Lee J Y. 2021. Alternative splicing of the basic helix-loop-helix transcription factor gene CmbHLH2 affects anthocyanin biosynthesis in ray florets of chrysanthemum(Chrysanthemum morifolium). Frontiers in Plant Science, 12:669315. doi: 10.3389/fpls.2021.669315 URL
[12]	Liu S, Zhang Y, Pan X, Li B, Yang Q, Yang C, Zhang J, Wu F, Yang A, Li Y. 2023. PIF1,a phytochrome-interacting factor negatively regulates drought tolerance and carotenoids biosynthesis in tobacco. International Journal of Biological Macromolecules, 247:125693. doi: 10.1016/j.ijbiomac.2023.125693 URL
[13]	Makkena S, Lamb R S. 2013. The bHLH transcription factor SPATULA regulates root growth by controlling the size of the root meristem. BMC Plant Biol, 13:1. doi: 10.1186/1471-2229-13-1 pmid: 23280064
[14]	Medina-Puche L, Martinez-Rivas F J, Molina-Hidalgo F J, Mercado J A, Moyano E, Rodriguez-Franco A, Caballero J L, Munoz-Blanco J, Blanco-Portales R. 2019. An atypical HLH transcriptional regulator plays a novel and important role in strawberry ripened receptacle. BMC Plant Biol, 19:586. doi: 10.1186/s12870-019-2092-4 pmid: 31881835
[15]	Meng Y, Wang Z, Wang Y, Wang C, Zhu B, Liu H, Ji W, Wen J, Chu C, Tadege M, Niu L, Lin H. 2019. The MYB activator WHITE PETAL1 associates with MtTT8 and MtWD40-1 to regulate carotenoid-derived flower pigmentation in Medicago truncatula. The Plant Cell, 31:2751-2767.
[16]	Ning G, Xiao X, Lv H, Li X, Zuo Y, Bao M. 2012. Shortening tobacco life cycle accelerates functional gene identification in genomic research. Plant Biology, 14:934-943. doi: 10.1111/j.1438-8677.2012.00571.x pmid: 23107371
[17]	Ohmiya A, Kishimoto S, Aida R, Yoshioka S, Sumitomo K. 2006. Carotenoid cleavage dioxygenase(CmCCD4a)contributes to white color formation in chrysanthemum petals. Plant Physiology, 142:1193-1201. pmid: 16980560
[18]	Qi Y, Zhou L, Han L, Zou H, Miao K, Wang Y. 2020. PsbHLH1,a novel transcription factor involved in regulating anthocyanin biosynthesis in tree peony(Paeonia suffruticosa). Plant Physiology and Biochemistry, 154:396-408. doi: 10.1016/j.plaphy.2020.06.015 URL
[19]	Qin J, Zhao C, Wang S, Gao N, Wang X, Na X, Wang X, Bi Y. 2022. PIF4-PAP1 interaction affects MYB-bHLH-WD40 complex formation and anthocyanin accumulation in Arabidopsis. Journal of Plant Physiology, 268:153558.
[20]	Sun Q, He Z, Feng D, Wei R, Zhang Y, Ye J, Chai L, Xu J, Cheng Y, Xu Q, Deng X. 2024. The abscisic acid-responsive transcriptional regulatory module CsERF110-CsERF53 orchestrates citrus fruit coloration. Plant Communications, 5:101065. doi: 10.1016/j.xplc.2024.101065 URL
[21]	Sun Q, He Z, Ye J, Wei R, Feng D, Zhang Y, Chai L, Cheng Y, Xu Q, Deng X. 2025a. A novel C2H2-type zinc-finger transcription factor, CitZAT4,regulates ethylene-induced orange coloration in Satsuma mandarin flavedo(Citrus unshiu Marc.). Journal of Integrative Plant Biology, 67:294-310. doi: 10.1111/jipb.v67.2 URL
[22]	Sun Q, He Z, Zhang Y, Feng D, Wei R, Chai L, Guo W, Xu J, Cheng Y, Xu Q, Ye J, Deng X. 2025b. CsbHLH6 positively regulates abscisic acid-mediated citrus fruit coloration during postharvest storage. Postharvest Biology and Technology, 219:113229. doi: 10.1016/j.postharvbio.2024.113229 URL
[23]	Tissot N, Robe K, Gao F, Grant-Grant S, Boucherez J, Bellegarde F, Maghiaoui A, Marcelin R, Izquierdo E, Benhamed M, Martin A, Vignols F, Roschzttardtz H, Gaymard F, Briat J F, Dubos C. 2019. Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3. New Phytologist, 223:1433-1446. doi: 10.1111/nph.2019.223.issue-3 URL
[24]	Verma R K, Kumar V V S., Yadav S K, Kumar T S, Rao M V, Chinnusamy V. 2020. Overexpression of Arabidopsis ICE 1 enhances yield and multiple abiotic stress tolerance in indica rice. Plant Signal & Behavior, 15:1814547.
[25]	Wang Y, Zhang C, Dong B, Fu J, Hu S, Zhao H. 2018. Carotenoid accumulation and its contribution to flower coloration of Osmanthus fragrans. Frontiers in Plant Science, 9:1499. doi: 10.3389/fpls.2018.01499 URL
[26]	Wang Ying, Zhang Chao, Fu Jianxin, Zhao Hongbo. 2016. Progresses on flower bud differentiation and flower opening in Osmanthus fragrans. Journal of Zhejiang A & F University, 33 (2):340-347. (in Chinese)
	王英, 张超, 付建新, 赵宏波. 2016. 桂花花芽分化和花开放研究进展. 浙江农林大学学报, 33 (2):340-347.
[27]	Yang T, Yao S, Hao L, Zhao Y, Lu W, Xiao K. 2016. Wheat bHLH-type transcription factor gene TabHLH1 is crucial in mediating osmotic stresses tolerance through modulating largely the ABA-associated pathway. Plant Cell Reports, 35:2309-2323. doi: 10.1007/s00299-016-2036-5 URL
[28]	Yue P, Jiang Z, Sun Q, Wei R, Yin Y, Xie Z, Larkin RM, Ye J, Chai L, Deng X. 2023. Jasmonate activates a CsMPK6-CsMYC2 module that regulates the expression of β-citraurin biosynthetic genes and fruit coloration in orange(Citrus sinensis). The Plant Cell, 35:1167-1185. doi: 10.1093/plcell/koac363 URL
[29]	Zang Dekui, Xiang Qibai. 2004. Studies on the cultivar classification of Chinese sweet osmanthus. Journal of Chinese Landscape Architecture,(11):43-52. (in Chinese)
	臧德奎, 向其柏. 2004. 中国桂花品种分类研究. 中国园林,(11):43-52.
[30]	Zhang J, Hu Z, Yao Q, Guo X, Nguyen V, Li F, Chen G. 2018. A tomato MADS-box protein,SlCMB1,regulates ethylene biosynthesis and carotenoid accumulation during fruit ripening. Scientific Reports, 8:3413. doi: 10.1038/s41598-018-21672-8
[31]	Zhou D, Shen Y, Zhou P, Fatima M, Lin J, Yue J, Zhang X, Chen LY, Ming R. 2019a. Papaya CpbHLH1/2 regulate carotenoid biosynthesis-related genes during papaya fruit ripening. Horticulture Research, 6:80. doi: 10.1038/s41438-019-0162-2
[32]	Zhou Q, Li Q, Li P, Zhang S, Liu C, Jin J, Cao P, Yang Y. 2019b. Carotenoid cleavage dioxygenases:identification,expression,and evolutionary analysis of this gene family in tobacco. International Journal of Molecular Sciences, 20 (22):5796. doi: 10.3390/ijms20225796 URL
[33]	Zhu Z, Chen G, Guo X, Yin W, Yu X, Hu J, Hu Z. 2017. Overexpression of SlPRE2,an atypical bHLH transcription factor,affects plant morphology and fruit pigment accumulation in tomato. Scientific Reports, 7:5786. doi: 10.1038/s41598-017-04092-y