Functional Analysis of the bZIP Transcription Factor CiFD in Regulating Lemon Branching

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

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (4): 1101-1112.doi: 10.16420/j.issn.0513-353x.2025-0507

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Functional Analysis of the bZIP Transcription Factor CiFD in Regulating Lemon Branching

YE Lixia¹^,², WU Yanmei², ZHANG Jinzhi²^,^*(), ZHANG Lei¹^,^*()

¹ Hubei Key Laboratory of Germplasm Innovation and Utilization of Fruit Trees，Institute of Fruit and Tea，Hubei Academy of Agricultural Science，Wuhan 430064，China
² College of Horticulture and Forestry Science，Huazhong Agricultural University，Wuhan 430070，China

Received:2025-07-23 Revised:2025-10-30 Online:2026-04-25 Published:2026-04-20
Contact: ZHANG Jinzhi, ZHANG Lei

Abstract

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

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.

Figures/Tables 8

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

Fig. 1 Phylogenetic analysis of CiFD gene

Fig. 2 Multiple sequence alignment of CiFD gene

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）.

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）

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）

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）

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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Functional Analysis of the bZIP Transcription Factor CiFD in Regulating Lemon Branching

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