柑橘CsBBX20转录因子调控花青苷积累的分子机制

doi:10.16420/j.issn.0513-353x.2026-0253

摘要/Abstract

摘要：

花青苷是决定柑橘果实着色的重要次生代谢物，受环境信号与多层级调控网络共同调控。以血橙果实为材料，克隆获得CsBBX20，阅读框长609 bp，编码202个氨基酸，含有2个高度保守的B-box结构域。在光照情况下，CsBBX20超量表达苹果愈伤系花青苷积累显著增加，而野生型苹果愈伤株系仅微量积累；在黑暗情况下，CsBBX20超表达系和野生型苹果愈伤均不积累花青苷。CsBBX20可直接结合CsF3’H、CsDFR、CsRuby1、CsHY5启动子并显著激活其表达，且CsBBX20与光信号关键因子CsHY5存在蛋白—蛋白互作，两者共表达对CsRuby1的激活效应显著增强。综上，CsBBX20可直接激活花青苷代谢关键基因，并与CsHY5形成复合体协同调控靶基因表达，促进光照条件下柑橘果实花青苷积累。

关键词: 柑橘, CsBBX20, 花青苷, 转录调控, CsHY5

Abstract:

Anthocyanins are important secondary metabolites responsible for citrus fruit coloration，and their accumulation is regulated by environmental signals and multilayered regulatory networks. In this study，CsBBX20 was cloned from blood orange fruit. The open reading frame of CsBBX20 is 609 bp，encoding a protein of 202 amino acids. Sequence analysis showed that CsBBX20 contains two highly conserved B-box domains. Functional analysis revealed that anthocyanin accumulation was significantly increased in CsBBX20-overexpressing apple calli under light conditions，whereas only trace amounts of anthocyanins accumulated in wild-type calli. However，under dark conditions，neither CsBBX20-overexpressing lines nor wild-type calli accumulated anthocyanins. Further biochemical analyses demonstrated that CsBBX20 can directly bind to the promoters of CsF3’H，CsDFR，CsRuby1，and CsHY5，and significantly activate their expression. Moreover，CsBBX20 physically interacts with the light signaling regulator CsHY5，and their co-expression markedly enhances the activation of CsRuby1. Taken together，CsBBX20 directly activates key genes involved in anthocyanin biosynthesis and forms a complex with CsHY5 to coordinately regulate the expression of target genes，thereby promoting anthocyanin accumulation in citrus fruit under light conditions.

Key words: citrus, CsBBX20, anthocyanin, transcriptional regulation, CsHY5

付佳玲, 魏钰镕, 孙娟, 韦幸, 杜林烛, 侯健琦, 刘宇琪, 徐强. 柑橘CsBBX20转录因子调控花青苷积累的分子机制[J]. 园艺学报, 2026, 53(7): 2075-2091.

FU Jialing, WEI Yurong, SUN Juan, WEI Xing, DU Linzhu, HOU Jianqi, LIU Yuqi, XU Qiang. CsBBX20 Regulates Anthocyanin Biosynthesis in Citrus[J]. Acta Horticulturae Sinica, 2026, 53(7): 2075-2091.

图/表 8

表1 本试验中所使用的引物

Table 1 Primers used in this study

用途 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequence
qRT-PCR	qPCR-CsActin	F：CCGACCGTATGAGCAAGGAAA
		R：TTCCTGTGGACAATGGATGGA
	qPCR-CsBBX20	F：TCAGTCTTTTGCTCAGCCGATG
		R：AAGCAAAGCACGTCTCTCCTGA
	qPCR-MdActin	F：TGACCGAATGAGCAAGGAAATTACT
		R：TACTCAGCTTTGGCAATCCACATC
	qPCR-MdCHS	F：GGAGACAACTGGAGAAGGACTGGAA
		R：CGACATTGATACTGGTGTCTTC
	qPCR-MdF3’H	F：TGGAAGCTTGTGAGGACTGGGGT
		R：CTCCTCCGATGGCAAATCAAAGA
	qPCR-MdDFR	F：GATAGGGTTTGAGTTCAAGTA
		R：TCTCCTCAGCAGCCTCAGTTTTCT
	qPCR-MdANS	F：CCAAGTGAAGCGGGTTGTGCT
		R：CAAAGCAGGCGGACAGGAGTAGC
	qPCR-MdMYB1	F：TGCCTGGACTCGAGAGGAAGACA
		R：CCTGTTTCCCAAAAGCCTGTGAA
	qPCR-MdHY5	F：TGAAGAGGTTGTTGAGGAA
		R：TTCTGCAAAGTGGAAAGC
基因克隆 Gene cloning	CsBBX20-CDS	F：ATGAAGATTTGGTGTGATGTGTGTG
		R：TCACAAATATAAAACATTAGTA
	CsHY5-CDS	F：ATGCAAGAACAAGCAACGAGTTC
		R：TTAAAAGGACCCATCGGCAT
超表达载体 Overexpression vector	Adapter attB1	F：GGGGACAAGTTTGTACAAAAAAGCAGGCT
	Adapter attB2	R：GGGGACCACTTTGTACAAGAAAGCTGGGT
	CsBBX20-Adpter	F：AAAAAGCAGGCTCCATGAAGATTTGGTGTGATGTGTGTG
		R：AGAAAGCTGGGTTTCACAAATATAAAACATTAGTA
	CsHY5-Adpter	F：AAAAAGCAGGCTCCATGCAAGAACAAGCAACGAGTTC
		R：AGAAAGCTGGGTTTTAAAAGGACCCATCGGCAT
LUC实验 Dual-luciferase reporter assay	CsRuby1pro-LUC	F：GGTGGAGATCGAATTCCATGGTGTCAAAGTCGGTGGCCACAAGG
	CsRuby1pro-LUC	R：TGTTTTTGGCGTCTTCCATGGAACTCCTAAGGAATCCGCCAT
	CsHY5pro-LUC	F：GGTGGAGATCGAATTCCATGGCCACCAAAGATAATAGCAA
	CsHY5pro-LUC	R：TGTTTTTGGCGTCTTCCATGGTTCTTCTTCTCCTATTTCT
蛋白表达实验 Protein expression assay	CsBBX20-pET32a	F：GCTGATATCGGATCCGAATTCATGAAGATTTGGTGTGATGTGTGTG
蛋白表达实验 Protein expression assay	CsBBX20-pET32a	R：TGCGGCCGCAAGCTTGTCGACCAAATATAAAACATTAGTAGATG
EMSA实验 Electrophoretic mobility shift assay	CsRuby1-wt	F：GCATGAAGTGAGGAGCACGTATTATTATACAAGCAG
		R：CTGCTTGTATAATAATACGTGCTCCTCACTTCATGC
	CsF3’H-wt	F：GTGGTTACGTACTTACACGTACACCCCTACCTGCAT
		R：ATGCAGGTAGGGGTGTACGTGTAAGTACGTAACCAC
	CsDFR-wt	F：AAACGGTCAGTTGATCACGTGGAGGGTAATTGGAGC
		R：GCTCCAATTACCCTCCACGTGATCAACTGACCGTTT
	CsHY5-wt	F：AAACAAGAGAGAATACAAGTGAGCTTTATAAAGATG
		R：CATCTTTATAAAGCTCACTTGTATTCTCTCTTGTTT
Y2H实验 Yeast two-hybrid assay	CsBBX20-AD	F：GCCATGGAGGCCAGTGAATTCATGAAGATTTGGTGTGATGTGT
		R：ACGATTCATCTGCAGCTCGAGTCACAAATATAAAACATTAGTA
	CsHY5-BD	F：AGGACCTGCATATGGCCATGGATGCAAGAACAAGCAACGAGTTC
		R：TGCAGGTCGACGGATCCCCGGGTTAAAAGGACCCATCGGCATTTG
LCI实验 Luciferase complementation imaging assay	CsBBX20-nLUC	F：ACGGGGGACGAGCTCGGTACCATGAAGATTTGGTGTGATGT
		R：CGCGTACGAGATCTGGTCGACCAAATATAAAACATTAGTAG
	CsHY5-cLUC	F：TACGCGTCCCGGGGCGGTACCATGCAAGAACAAGCAACGAGTTC
		R：ACGAAAGCTCTGCAGGTCGACTTAAAAGGACCCATCGGCATTTG

表1 本试验中所使用的引物

Table 1 Primers used in this study

用途 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequence
qRT-PCR	qPCR-CsActin	F：CCGACCGTATGAGCAAGGAAA
		R：TTCCTGTGGACAATGGATGGA
	qPCR-CsBBX20	F：TCAGTCTTTTGCTCAGCCGATG
		R：AAGCAAAGCACGTCTCTCCTGA
	qPCR-MdActin	F：TGACCGAATGAGCAAGGAAATTACT
		R：TACTCAGCTTTGGCAATCCACATC
	qPCR-MdCHS	F：GGAGACAACTGGAGAAGGACTGGAA
		R：CGACATTGATACTGGTGTCTTC
	qPCR-MdF3’H	F：TGGAAGCTTGTGAGGACTGGGGT
		R：CTCCTCCGATGGCAAATCAAAGA
	qPCR-MdDFR	F：GATAGGGTTTGAGTTCAAGTA
		R：TCTCCTCAGCAGCCTCAGTTTTCT
	qPCR-MdANS	F：CCAAGTGAAGCGGGTTGTGCT
		R：CAAAGCAGGCGGACAGGAGTAGC
	qPCR-MdMYB1	F：TGCCTGGACTCGAGAGGAAGACA
		R：CCTGTTTCCCAAAAGCCTGTGAA
	qPCR-MdHY5	F：TGAAGAGGTTGTTGAGGAA
		R：TTCTGCAAAGTGGAAAGC
基因克隆 Gene cloning	CsBBX20-CDS	F：ATGAAGATTTGGTGTGATGTGTGTG
		R：TCACAAATATAAAACATTAGTA
	CsHY5-CDS	F：ATGCAAGAACAAGCAACGAGTTC
		R：TTAAAAGGACCCATCGGCAT
超表达载体 Overexpression vector	Adapter attB1	F：GGGGACAAGTTTGTACAAAAAAGCAGGCT
	Adapter attB2	R：GGGGACCACTTTGTACAAGAAAGCTGGGT
	CsBBX20-Adpter	F：AAAAAGCAGGCTCCATGAAGATTTGGTGTGATGTGTGTG
		R：AGAAAGCTGGGTTTCACAAATATAAAACATTAGTA
	CsHY5-Adpter	F：AAAAAGCAGGCTCCATGCAAGAACAAGCAACGAGTTC
		R：AGAAAGCTGGGTTTTAAAAGGACCCATCGGCAT
LUC实验 Dual-luciferase reporter assay	CsRuby1pro-LUC	F：GGTGGAGATCGAATTCCATGGTGTCAAAGTCGGTGGCCACAAGG
	CsRuby1pro-LUC	R：TGTTTTTGGCGTCTTCCATGGAACTCCTAAGGAATCCGCCAT
	CsHY5pro-LUC	F：GGTGGAGATCGAATTCCATGGCCACCAAAGATAATAGCAA
	CsHY5pro-LUC	R：TGTTTTTGGCGTCTTCCATGGTTCTTCTTCTCCTATTTCT
蛋白表达实验 Protein expression assay	CsBBX20-pET32a	F：GCTGATATCGGATCCGAATTCATGAAGATTTGGTGTGATGTGTGTG
蛋白表达实验 Protein expression assay	CsBBX20-pET32a	R：TGCGGCCGCAAGCTTGTCGACCAAATATAAAACATTAGTAGATG
EMSA实验 Electrophoretic mobility shift assay	CsRuby1-wt	F：GCATGAAGTGAGGAGCACGTATTATTATACAAGCAG
		R：CTGCTTGTATAATAATACGTGCTCCTCACTTCATGC
	CsF3’H-wt	F：GTGGTTACGTACTTACACGTACACCCCTACCTGCAT
		R：ATGCAGGTAGGGGTGTACGTGTAAGTACGTAACCAC
	CsDFR-wt	F：AAACGGTCAGTTGATCACGTGGAGGGTAATTGGAGC
		R：GCTCCAATTACCCTCCACGTGATCAACTGACCGTTT
	CsHY5-wt	F：AAACAAGAGAGAATACAAGTGAGCTTTATAAAGATG
		R：CATCTTTATAAAGCTCACTTGTATTCTCTCTTGTTT
Y2H实验 Yeast two-hybrid assay	CsBBX20-AD	F：GCCATGGAGGCCAGTGAATTCATGAAGATTTGGTGTGATGTGT
		R：ACGATTCATCTGCAGCTCGAGTCACAAATATAAAACATTAGTA
	CsHY5-BD	F：AGGACCTGCATATGGCCATGGATGCAAGAACAAGCAACGAGTTC
		R：TGCAGGTCGACGGATCCCCGGGTTAAAAGGACCCATCGGCATTTG
LCI实验 Luciferase complementation imaging assay	CsBBX20-nLUC	F：ACGGGGGACGAGCTCGGTACCATGAAGATTTGGTGTGATGT
		R：CGCGTACGAGATCTGGTCGACCAAATATAAAACATTAGTAG
	CsHY5-cLUC	F：TACGCGTCCCGGGGCGGTACCATGCAAGAACAAGCAACGAGTTC
		R：ACGAAAGCTCTGCAGGTCGACTTAAAAGGACCCATCGGCATTTG

图1 CsBBX20的系统发育树 AT：拟南芥；Cs：柑橘；Fv：草莓；Cp：木瓜；Sl：番茄；Pp：桃；Pa：樱桃；Pb：梨；Md：苹果。下同

Fig. 1 Phylogenetic tree analysis of CsBBX20 At：Arabidopsis thaliana；Cs：Citrus sinensis；Fv：Fragaria vesca subsp. vesca；Cp：Carica papaya；Sl：Solanum lycopersicum；Pp：Prunus persica；Pa：Prunus avium；Pb：Pyrus × bretschneideri；Md：Malus × domestica. The same below

图2 不同物种BBX20的蛋白序列比对灰色填充表示氨基酸序列相似度 > 75%

Fig. 2 Multiple sequence alignment of BBX20 protein sequences from different species Gray shading indicates amino acid sequence similarity greater than 75%

图3 超表达CsBBX20苹果愈伤系的表型分析 A：超表达CsBBX20苹果愈伤的鉴定，明场表示自然光照下的愈伤形态，GFP表示滤光片下的愈伤的荧光信号；B：光照和黑暗条件下野生型（WT）及CsBBX20（35S：CsBBX20）苹果愈伤的表型；C、E：光照（C）和黑暗（E）条件下野生型及CsBBX20苹果愈伤中CsBBX20的表达情况；D、F：光照（D）和黑暗（F）条件下野生型及CsBBX20苹果愈伤中花青苷的积累情况。WT表示野生型，35S：CsBBX20表示CsBBX20超表达系，OE#1、OE#2和OE#3表示CsBBX20超表达系#1，#2，#3。Student’s t-test，* P < 0.05，** P < 0.01，*** P < 0.001。下同

Fig. 3 Phenotypic characterization of CsBBX20-overexpressing apple calli A：Identification of CsBBX20-overexpressing apple calli，Bright-field images show the morphology of apple calli under natural light conditions，whereas GFP images represent calli observed under a fluorescence filter；B：Phenotypes of wild-type and CsBBX20-overexpressing apple calli under light and dark conditions；C，E：Expression levels of CsBBX20 in wild-type and CsBBX20-overexpressing apple calli under light（C）conditions and dark（E）conditions；D，F：Anthocyanin accumulation in wild-type and CsBBX20-overexpressing apple calli under both light（D）and dark（F）conditions. WT denotes the wild type，35S：CsBBX20 indicates CsBBX20-overexpressing lines，OE#1，OE#2，and OE#3 represent three independent CsBBX20-overexpressing lines. Student’s t-test，* P < 0.05，** P < 0.01，*** P < 0.001. The same below

图4 光照（A）和黑暗（B）条件下超表达CsBBX20苹果愈伤系的花青苷通路关键基因表达情况

Fig. 4 Expression of key genes in the anthocyanin biosynthetic pathway in CsBBX20-overexpressing apple calli under light（A）and dark（B）conditions

图5 CsBBX20对CsRuby1、CsF3’H和CsDFR启动子的调控分析 A：CsRuby1、CsF3’H和CsDFR启动子上G-box元件展示；B：烟草双荧光素酶实验用到的载体模式图；C：烟草双荧光素酶实验验证CsBBX20对CsRuby1、CsF3’H和CsDFR启动子的激活情况，EV表示pk7WG2D空载；D：EMSA实验验证CsBBX20-His对CsRuby1、CsF3’H和CsDFR启动子的G-box元件的结合情况。+和-符号分别表示蛋白质或探针的添加和缺失；WT探针使用生物素标记（Bio labeled）

Fig. 5 Analysis of CsBBX20 regulation on the promoters of CsRuby1，CsF3’H and CsDFR A：Distribution of G-box cis-elements in the promoters of CsRuby1，CsF3’H，and CsDFR；B：Schematic representation of the constructs used in the dual-luciferase assay in Nicotiana benthamiana；C：Dual-luciferase assay validating the activation of CsRuby1，CsF3’H，and CsDFR promoters by CsBBX20，EV：pk7WG2D empty vector；D：EMSA showing the binding of CsBBX20-His to the G-box elements in the promoters of CsRuby1，CsF3’H，and CsDFR.“+”and“-”indicate the presence or absence of protein or probe，respectively. Wild-type probes were biotin-labeled（Bio-labeled）

图6 CsBBX20和CsHY5的互作关系分析 A：酵母双杂交实验（Y2H）；B：烟草荧光素酶互补实验（LCI）（a表示nLUC + cLUC组合，b表示CsBBX20-nLUC + cLUC组合，c表示nLUC + CsHY5-cLUC组合，d表示CsBBX20-nLUC + CsHY5-cLUC组合）；C：EMSA实验。+和-符号分别表示蛋白质或探针的添加和缺失，WT探针使用生物素标记（Bio labeled），竞争探针（Competitor）和突变探针（Mut probe）未标记；D：烟草双荧光素酶实验（LUC）；E：烟草LUC实验。1表示EV + CsRuby1pro组合，2表示EV + CsHY5 + CsRuby1pro组合，3表示EV + CsBBX20 + CsRuby1pro组合，4表示CsBBX20 + CsHY5 + CsRuby1pro组合

Fig. 6 Analysis of the interaction between CsBBX20 and CsHY5 A：Yeast two-hybrid（Y2H）；B：Luciferase complementation imaging assay（LCI）（a indicated the nLUC + cLUC combination，b indicates the CsBBX20-nLUC + cLUC combination，c indicates the nLUC + CsHY5-cLUC combination，d indicates the CsBBX20-nLUC + CsHY5-cLUC combination）；C：Electrophoretic Mobility Shift Assay.“+”and“-”indicate the presence or absence of protein or probe，respectively，the WT probe was biotin-labeled，whereas the competitor（Competitor）and mutant probe（Mut probe）were unlabeled；D：Dual-Luciferase Reporter Assay in tobacco leaves；E：Tobacco LUC assay. 1 indicates the EV + CsRuby1pro combination，2 indicates EV + CsHY5 + CsRuby1pro，3 indicates EV + CsBBX20 + CsRuby1pro，4 indicates CsBBX20 + CsHY5 + CsRuby1pro

图7 光照和低温处理下CsBBX20的表达变化情况

Fig. 7 Expression patterns of CsBBX20 under light and low-temperature treatments

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