荔枝LcTFL1基因的克隆与功能分析

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

摘要/Abstract

摘要：

目的 TFL1基因是植物开花途径中的重要调控基因，克隆荔枝LcTFL1基因并构建系统发育树，分析其表达模式并进行功能研究，为荔枝成花调控提供靶标基因。方法基于前期获取的转录组数据，通过设计特异性引物，利用PCR技术从‘妃子笑’荔枝叶片中克隆得到LcTFL1基因，结合生物信息学分析其蛋白保守结构和系统进化关系，通过定量PCR技术选取荔枝不同组织，明确该基因的组织表达模式，利用亚细胞定位确定其蛋白表达位置，通过构建植物双源表达载体，异源过表达拟南芥，统计开花时间表型，分析验证LcTFL1基因功能。结果 LcTFL1基因编码区全长为513 bp，编码170个氨基酸，为亲水性蛋白，具有保守PEBP家族结构域，启动子区域存在多个响应光、激素和胁迫的顺式作用元件，亚细胞定位显示其蛋白定位在细胞质中。系统进化关系表明荔枝LcTFL1基因和同科的文冠果（Xanthoceras sorbifolium）的JRO89相似性最高。组织表达分析显示，LcTFL1在种子的表达量最高，其次为顶芽和果实。在拟南芥中过表达LcTFL1基因，过表达拟南芥植株表现为始花期莲座叶片数量显著增多，叶片长度和宽度显著降低，且植株出现晚花表型。结论 LcTFL1高表达会延迟拟南芥开花，作为调控开花的抑制因子存在。

关键词: 荔枝, LcTFL1基因, 表达模式, 过表达, 开花

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

史发超, 姜永华, 刘海伦, 文英杰, 严倩. 荔枝LcTFL1基因的克隆与功能分析[J]. 生物技术通报, 2025, 41(9): 159-167.

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.

图/表 7

表1 引物列表

Table 1 Primer list

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

图1 LcTFL1蛋白生物信息学分析A：蛋白亲水/疏水性预测；B：蛋白二级结构预测；C：蛋白三级结构预测；D：蛋白序列比对

Fig. 1 Bioinformation analysis of LcTFL1A: Hydrophilic and hydrophobic prediction of protein. B: Secondary structure prediction of protein. C: Tertiary structure prediction of protein. D: Protein sequence alignment

表2 LcTFL1启动子调控元件分析

Table 2 Regulatory element analysis of LcTFL1 promoter

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

图2 LcTFL1蛋白的系统进化树分析

Fig. 2 Phylogenetic tree analysis of LcTFL1 protein

图3 荔枝LcTFL1不同组织表达分析不同字母表示差异显著（P<0.05）

Fig. 3 Expressions of LcTFL1 gene in different tissues in litchi (Litchi chinensis Sonn.)Different letters indicate significant difference (P<0.05)

图4 LcTFL1的亚细胞定位pBWA（V）HS-TFL1--GLosgfp在拟南芥原生质体中表达为绿色荧光蛋白，细胞核marker表达为红色荧光蛋白，紫色为叶绿体在特定波长下显示的紫色荧光

Fig. 4 Subcellular localization of LcTFL1The pBWA(V)HS-TFL1--GLosgfp is expressed as green fluorescent proteinin Arabidopsis thaliana protoplasts, the nuclear marker is expressed as red fluorescent protein, and purple is the purple fluorescence displayed by chloroplasts under specific wavelengths

图5 拟南芥转基因植株表型A、B：拟南芥野生型与过表达植株开花早晚形态；C：叶片数量及形态；D：叶片数量；E：叶片长度；F：叶片宽度。*代表差异显著（P<0.05），WT 代表野生型，OE（4-2）、OE（3-1）、OE（4-1）代表转基因株系

Fig. 5 Plant phenotype of transgenic ArabidopsisA, B: Phenotypic in tansgenic and wild-type Arabidopsis of flowering time. C: Number and morphology of leaves at bolting. D: Leaf number at bolting. E: Leaf length. F: Leaf width. * indicates significant difference at P<0.05. WT indicates wild type, OE (4-2), OE (3-1) and OE (4-1) indicate transgenic lines

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