Cloning and Flowering Regulation Mechanism of BoFLC3 in Broccoli

doi:10.13560/j.cnki.biotech.bull.1985.2025-1354

Abstract

Abstract:

Objective FLOWERING LOCUS C (FLC) is one of the key genes regulating flowering in plants. By cloning BoFLC3 from broccoli and characterizing its function, this study aimed to provide insights into the molecular regulatory mechanism underlying the floral transition in broccoli. Method Using broccoli as the experimental material, BoFLC3 was cloned by homology-based cloning. Its sequence characteristics, conserved domains, and phylogenetic relationships were analyzed using bioinformatics tools. The expression profiles of BoFLC3 in different tissues and under various treatments were detected by RT-qPCR to determine its expression specificity. A subcellular localization vector was constructed and transiently overexpressed in tobacco to determine the subcellular localization of the BoFLC3 protein. Furthermore, BoFLC3 was heterologously overexpressed in Arabidopsis thaliana, and its role in flowering was analyzed by comparing bolting and flowering phenotypes between transgenic and wild-type plants. Result The full-length cDNA of BoFLC3 is 594 bp, encoding a protein of 197 amino acids. The deduced BoFLC3 protein contains a typical MADS domain and a K-box domain and was localized in the nucleus. Phylogenetic analysis showed that BoFLC3 shares high homology with FLC from cabbage (Brassica oleracea var. capitata). Expression pattern analysis revealed that BoFLC3 transcript levels were relatively high in mature leaves and shoot tips, and significantly decreased in shoot tips after low-temperature treatment. Heterologous overexpression of BoFLC3 in A. thaliana significantly delayed bolting and flowering time. In the overexpression lines, the transcript levels of the flowering-promoting genes AtFT, AtLFY, AtAP1, and AtSPL were downregulated, whereas the expression of the flowering repressor gene AtSVP was upregulated. Conclusion BoFLC3 plays a critical role in the flowering process. Its heterologous overexpression significantly delayed bolting and flowering, indicating that this gene functions as a negative regulator in the floral induction pathway.

Key words: broccoli, BoFLC3, expression pattern, genetic transformation, flowering

ZHANG Jing, LI Yuan-yuan, WANG Yan, JIANG Ya-jie, YANG Han-bing, CHAI Wen-chen, HUO Chen-si, LUO Dan, YAN Shi-jiang. Cloning and Flowering Regulation Mechanism of BoFLC3 in Broccoli[J]. Biotechnology Bulletin, 2026, 42(9): 169-177.

Figures/Tables 7

Table 1 Phenotypic analysis of Arabidopsis plants overexpressing BoFLC3 at T3 generation

植株

Group

抽薹时间

Bolting time （d）

莲座叶片数

Number of leaves

初花时间

Time to first flowering （d）

初花期株高

Plant height at first flowering （cm）

初花期花薹粗

Flower stalk diameter at first flowering （mm）

Fig. 1 Cloning and deduced amino acid sequence of BoFLC3A: PCR amplification of BoFLC3 (M: 2 000 bp DNA marker; 1: BoFLC3). B: The nucleotide and amino acid sequences of BoFLC3

Fig. 2 Sequence alignment and phylogenetic analysis of BoFLC3 proteinA: Sequence alignment of BoFLC3 with homologous FLC proteins from diverse species. B: Phylogenetic tree of BoFLC3 and related FLC proteins. Red box indicates the species used in this study

Fig. 3 Tissue expression pattern of BoFLC3 in broccoliCK: Control plant grown at 25 ℃/17 ℃. V15: Plants treated at 17 ℃/9 ℃ for 15 d. * P<0.05, ** P<0.01

Fig. 4 Subcellular localization of BoFLC3

Fig. 5 Identification of transgenic Arabidopsis plantsA: PCR verification of BoFLC3 in transgenic Arabidopsis plants (M: 2 000 bp DNA marker, 1: pBI121-BoFLC3 plasmid (positive control), 2: wild-type (negative control), 3‒4: transgenic Arabidopsis plants). B: The relative expression level of BoFLC3 in transgenic plants. C: Phenotypes of BoFLC3 overexpression transgenic Arabidopsis plants. WT: Wild type. OE: BoFLC3 overexpression lines. Different letters indicate significant differences at P<0.05. The same below

Fig. 6 Effects of BoFLC3 overexpression on the expression of flowering-related genes in Arabidopsis

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