bZIP转录因子CiFD调控柠檬分枝的功能解析

doi:10.16420/j.issn.0513-353x.2025-0507

摘要/Abstract

摘要：

以柠檬为材料对bZIP转录因子CiFD进行系统进化分析，发现该蛋白与拟南芥A类bZIP转录因子AtbZIP27亲缘关系最近。CiFD在茎中的表达水平显著高于其他器官，且在有分枝的茎中的表达水平显著高于没有分枝的。在烟草和枳中对CiFD基因进行过表达，转基因株系分枝数显著多于未转基因对照。定量分析发现，CiFD转基因烟草和枳中TCP转录因子THORNIDENTITY1（TI1）的烟草同源基因NtTI1和枳同源基因PtTI1表达水平均显著下调。序列分析发现，CiTI1启动子包含bZIP转录因子结合元件G-box。酵母单杂实验表明，CiFD可以与CiTI1启动子结合，双荧光素酶实验进一步证实，CiFD可抑制CiTI1基因表达。这些结果揭示了CiFD通过负调控CiTI1表达参与调控柠檬分枝。

关键词: 柑橘, CiFD, CiTI1, 基因, 分枝

Abstract:

In lemon，phylogenetic analysis of the bZIP transcription factor CiFD revealed that it is most closely related to the Arabidopsis thaliana class A bZIP transcription factor AtbZIP27. The expression level of the CiFD gene was significantly higher in stems compared to other organs，and higher in stems with branches than those without branches. Overexpression of the CiFD gene in tobacco and trifoliate orange showed that the number of branches in transgenic plants was significantly higher than that in the non-transgenic control. Additionally，the qRT-PCR analysis revealed that the expression levels of NtTI1 and CiTI1，the homologous genes of TCP transcription factor THORNIDENTITY1（TI1），were significantly down-regulated in CiFD transgenic tobacco and trifoliate orange. Sequence analysis revealed that the CiTI1 promoter contains a bZIP transcription factor binding element，G-box. Yeast one-hybrid experiments showed that CiFD could bind to the CiTI1 promoter，and Dual-luciferase assays further indicated that CiFD protein represses CiTI1 gene expression. These findings indicate that the CiFD gene participates in regulating lemon branching by inhibiting the expression of the CiTI1 gene.

Key words: citrus, CiFD, CiTI1, gene, branching

叶丽霞, 吴燕美, 张金智, 张蕾. bZIP转录因子CiFD调控柠檬分枝的功能解析[J]. 园艺学报, 2026, 53(4): 1101-1112.

YE Lixia, WU Yanmei, ZHANG Jinzhi, ZHANG Lei. Functional Analysis of the bZIP Transcription Factor CiFD in Regulating Lemon Branching[J]. Acta Horticulturae Sinica, 2026, 53(4): 1101-1112.

图/表 8

表1 基因克隆、表达及载体构建所用引物序列

Table 1 Primers sequence of gene cloning，expression and vector construction

用途 Application	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因克隆 Gene clone	CiFD-cDNA-F	ATGTGGCCATCTCCAGCTAGAAACA
基因克隆 Gene clone	CiFD-cDNA-R	TTATTCTTACTTCTCAAAATGGAGC
启动子克隆 Promoter clone	pCiTI1-F	GCTGTTATGTGATATATCCCT
启动子克隆 Promoter clone	pCiTI1-R	AGCCATCTTTTTCTTAATCTTTC
基因过表达 Gene overexpression	CiFD-GFP-F	GCCCGTCGACCCCGGGGGTACCATGTGGCCATCTCCAGCTAG
基因过表达 Gene overexpression	CiFD-GFP-R	CGCCCTTGCTCACCATGAATTCAAATGGAGCAGTTGAGGTTC
酵母单杂 Yeast one-hybrid	pAbAi-pCiTI1-F	ATGATGAATTGAAAAGCTTGCTGTTATGTGATATATCCCT
酵母单杂 Yeast one-hybrid	AD-CiFDɑ-R	CTACGATTCATCTGCAGCTCGAGTCAAAATGGAGCACTTGAGG
双荧光素酶 Dual-luciferase	CiFD-62SK-F	GCCGCTCTAGAACTAGTGGATCCATGTGGCCATCTCCAGCTAG
	CiFD-62SK-R	GAGGTCGACGGTATCGATAAGCTTTCAAAATGGAGCAGTTGAGG
	CiTI1-0800-F	AGGTCGACGGTATCGATAAGCTTGCTGTTATGTGATATATCCCT
	CiTI1-0800-R	GCCGCTCTAGAACTAGTGGATCCAGCCATCTTTTTCTTAATCTTTC
基因定量 Gene quantification	CiFD-F	ATCACGCTAACACTGCCATCC
	CiFD-R	CAGACCCAGAATTCAGGCTC
	PtTI1-F	CTTCAAAGAAAGACAGGCATAG
	PtTI1-R	ACCAACATCGCCACAGCA
	NtTI1-F	GCGCTACTGCTAATATTGGTGC
	NtTI1-R	TCGCTCTAGCTTGCTTTCTTGA

图1 CiFD基因的系统进化分析

Fig. 1 Phylogenetic analysis of CiFD gene

图2 CiFD多重序列比对

Fig. 2 Multiple sequence alignment of CiFD gene

图3 CiFD基因的时空表达分析不同小写字母表示在P < 0.05水平差异显著（Duncan新复极差法）；**表示在P < 0.01水平差异极显著（独立样本t检验）

Fig. 3 Spatio-temporal expression analysis of CiFD gene Different lowercase letters indicate significant differences at the P < 0.05 level（Duncan's new multiple range test）；** indicates highly significant differences at the P < 0.01 level（Independent samples t-test）.

图4 CiFD基因超表达烟草分枝表型（A ~ C）及分枝数统计（D）对照：烟草野生型；CiFD-2#、CiFD-4#和CiFD-5#：CiFD基因超表达烟草株系。红色箭头指示有无分枝的主茎。采用独立样本t检验进行显著性分析，***表示差异极显著水平（P < 0.001）

Fig. 4 Branching phenotype（A-C）and branch number statistics（D）of CiFD gene overexpressing tobacco Control：Tobacco wild type；CiFD-2#，CiFD-4#，and CiFD-5#：CiFD gene overexpression tobacco lines. The position of the red arrow indicates the main stem with or without branches. Independent sample t-test was used for significance analysis，and ***indicated that the difference was extremely significant（P < 0.001）

图5 CiFD基因超表达枳分枝表型（A ~ C）及分枝数统计（D）对照：枳野生型；CiFD-OE1、CiFD-OE15和CiFD-OE24：CiFD基因超表达枳株系。红色箭头指示有无分枝的主茎。采用独立样本t检验进行显著性分析，*表示差异显著水平（P < 0.05），***表示差异极显著水平（P < 0.001）

Fig. 5 Branching phenotype（A-C）of the CiFD gene overexpression trifoliate orange and branch number statistics（D） Control：Wild type control；CiFD-OE1，CiFD-OE15，and CiFD-OE24：Three CiFD gene overexpressing Poncirus trifoliata lines. The position of the red arrow indicates the main stem with or without branches. The independent sample t-test was used for significance analysis. * indicated a significant level of difference（P < 0.05），and *** indicated a very significant level of difference（P < 0.001）

图6 烟草TI1和枳TI1基因在CiFD基因超表达植株中的表达水平分析采用独立样本t检验进行显著性分析，**表示差异显著水平（P < 0.01），***表示差异极显著水平（P < 0.001）

Fig. 6 Analysis of the expression levels of the NtTI1 gene and the PtTI1 gene in CiFD gene overexpressing plants The independent sample t-test was used for significance analysis. ** indicated a significant level of difference（P < 0.01），and *** indicated a very significant level of difference（P < 0.001）

图7 酵母单杂交（A）及双荧光素酶（B）实验验证CiFD蛋白与CiTI1基因启动子结合，抑制其表达 *** P < 0.001

Fig. 7 Yeast one-hybrid（A）and dual luciferase（B）experiments verified that CiFD protein binds to the CiTI1 gene promoter and inhibits its expression

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