Cloning and Functional Analysis of LcTFL1 Gene in Litchi chinensis Sonn.

doi:10.13560/j.cnki.biotech.bull.1985.2025-0147

Abstract

Abstract:

Objective The TERMINAL FLOWER 1 (TFL1) gene is a crucial regulator in the plant flowering pathway. Our study aims to clone the LcTFL1 gene of litchi (Litchi chinensis Sonn.) and analyze its expression pattern and function, with the goal of identifying a marker gene for litchi flower formation regulation. This helps unlock the genetic mechanisms governing litchi floral development. Method Using previously obtained transcriptome data, we designed specific primers and successfully cloned the LcTFL1 gene from 'Feizixiao' litchi leaves by using the PCR technology. To verify its function, we used bioinformatics for protein structure and phylogenetic analysis. Through quantitative PCR technology, different tissues of litchi were selected to clarify the tissue expression pattern of this gene. Subcellular localization was determined by fusing LcTFL1 with a fluorescent tag and observing with confocal microscopy. By constructing a plant dual-source expression vector, we also overexpressed LcTFL1 in Arabidopsis thaliana to study its impact on flowering. Result The full-length coding region of LcTFL1 is 513 bp, which encodes 170 amino acids. It is a hydrophilic protein with a conserved PEBP domain. The promoter has cis-acting elements responsive to light, hormones, and stress. LcTFL1 localizes to the cytoplasm. Phylogenetically, it is the closest to JRO89 of Xanthoceras sorbifolium. Its expression is the highest in seeds, followed by apical buds and fruits. When the LcTFL1 gene was overexpressed in A. thaliana, the overexpressed A. thaliana plants showed that the number of rosette leaves at the initial flowering stage significantly increased, the length and width of leaves significantly reduced, and the plants demonstrated a late-flowering phenotype. Conclusion High expression of LcTFL1 delays A. thaliana flowering, indicating it acts as an inhibitory factor in flowering regulation. This discovery has implications for understanding plant development and for crop improvement in litchi.

Key words: litchi, LcTFL1 gene, expression pattern, over-expression, flowering

SHI Fa-chao, JIANG Yong-hua, LIU Hai-lun, WEN Ying-jie, YAN Qian. Cloning and Functional Analysis of LcTFL1 Gene in Litchi chinensis Sonn.[J]. Biotechnology Bulletin, 2025, 41(9): 159-167.

Figures/Tables 7

References 39

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用途 Usage	引物名称 Prime name	序列 Sequence （5′-3′）
克隆 Clone	LcTFL1-F	ATGACAGAAACACTATCT
克隆 Clone	LcTFL1-R	TCATCTTCTTCTAGCAGC
定位 Location	LcTFL1-F	CAGTGGTCTCACAACATGACAGAAACACTATCTGTGGGAAGAGTTG
定位 Location	LcTFL1-R	CAGTGGTCTCATACATCTTCTTCTAGCAGCGGTTTCTCTTTGG
过表达 Over-expression	LcTFL1-F	AACACGGGGGACTTTGCAACATGACAGAAACACTATC
过表达 Over-expression	LcTFL1-R	CCGTCGTGGTCTTTGTAATCTCTTCTTCTAGCAGCGG
内参 Actin GAPDH	Actin-F	ACCGTATGAGCAAGGAAATCACTG
内参 Actin GAPDH	Actin-R	TCGTCGTACTCACCCTTTGAAATC
荧光定量 RT-qPCR	LcTFL1-F	ATGGCCTCAGAGCTGCTTAT
荧光定量 RT-qPCR	LcTFL1-R	GTGGTGCCAGGAATGTTTGT

用途 Usage	引物名称 Prime name	序列 Sequence （5′-3′）
克隆 Clone	LcTFL1-F	ATGACAGAAACACTATCT
克隆 Clone	LcTFL1-R	TCATCTTCTTCTAGCAGC
定位 Location	LcTFL1-F	CAGTGGTCTCACAACATGACAGAAACACTATCTGTGGGAAGAGTTG
定位 Location	LcTFL1-R	CAGTGGTCTCATACATCTTCTTCTAGCAGCGGTTTCTCTTTGG
过表达 Over-expression	LcTFL1-F	AACACGGGGGACTTTGCAACATGACAGAAACACTATC
过表达 Over-expression	LcTFL1-R	CCGTCGTGGTCTTTGTAATCTCTTCTTCTAGCAGCGG
内参 Actin GAPDH	Actin-F	ACCGTATGAGCAAGGAAATCACTG
内参 Actin GAPDH	Actin-R	TCGTCGTACTCACCCTTTGAAATC
荧光定量 RT-qPCR	LcTFL1-F	ATGGCCTCAGAGCTGCTTAT
荧光定量 RT-qPCR	LcTFL1-R	GTGGTGCCAGGAATGTTTGT

元件类型 Type	相关元件 Structure	数量 Number	序列 Sequence	功能注释 Function annotation
光响应元件 Light response element	TCT-motif	1	TCTTAC	Part of a light responsive element
	TCCC-motif	1	TCTCCCT	Part of a light responsive element
	GATA-motif	1	AAGGATAAGG	Part of a light responsive element
	G-box	1	CACGTC	Part of a light responsive element
	G-Box	1	CACGTT	Part of a light responsive element
	Box 4	3	ATTAAT	Part of a light responsive element
	ARE	1	AAACCA	Part of a light responsive element
	ACE	1	GACACGTATG	Part of a light responsive element
	circadian	1	CAAAGATATC	Involved in circadian control
激素响应元件 Hormone response element	ABRE	2	ACGTG	In the abscisic acid responsive
胁迫响应元件Stress response element	TC-rich repeats	2	GTTTTCTTAC	Involved in defense and stress responsiveness
胁迫响应元件Stress response element	ARE	1	AAACCA	Anaerobic responsive element

元件类型 Type	相关元件 Structure	数量 Number	序列 Sequence	功能注释 Function annotation
光响应元件 Light response element	TCT-motif	1	TCTTAC	Part of a light responsive element
	TCCC-motif	1	TCTCCCT	Part of a light responsive element
	GATA-motif	1	AAGGATAAGG	Part of a light responsive element
	G-box	1	CACGTC	Part of a light responsive element
	G-Box	1	CACGTT	Part of a light responsive element
	Box 4	3	ATTAAT	Part of a light responsive element
	ARE	1	AAACCA	Part of a light responsive element
	ACE	1	GACACGTATG	Part of a light responsive element
	circadian	1	CAAAGATATC	Involved in circadian control
激素响应元件 Hormone response element	ABRE	2	ACGTG	In the abscisic acid responsive
胁迫响应元件Stress response element	TC-rich repeats	2	GTTTTCTTAC	Involved in defense and stress responsiveness
胁迫响应元件Stress response element	ARE	1	AAACCA	Anaerobic responsive element