青花菜BoFLC3的克隆与开花调控机制研究

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

摘要/Abstract

摘要：

目的 FLC是调控植物开花的关键基因之一，通过克隆青花菜BoFLC3，并对其功能进行研究，以期为阐明青花菜开花转变的分子调控机制提供理论依据。方法以青花菜为材料，同源克隆获得BoFLC3，通过生物信息学工具分析其序列特性、保守结构域及系统发育关系；采用RT-qPCR技术检测BoFLC3在不同组织及不同处理下的表达谱，明确其表达特异性；构建亚细胞定位载体进行烟草瞬时过表达，明确其蛋白的细胞定位情况；进一步在拟南芥中异源过表达BoFLC3，并通过对比转基因株系与野生型植株的抽薹和开花表型差异，分析该基因在开花过程中的作用。结果 BoFLC3的cDNA全长为594 bp，编码197个氨基酸，其编码蛋白含有典型的MADS结构域和K-box结构域，蛋白定位于细胞核。系统进化分析表明，青花菜FLC3与结球甘蓝FLC的同源性较高。表达模式分析显示，BoFLC3在成熟叶和茎尖中表达水平较高，且在低温处理后的青花菜茎尖中表达量显著下降。在拟南芥中异源过表达BoFLC3可显著推迟植株的抽薹及开花时间，过表达株系中开花促进基因AtFT、AtLFY、AtAP1和AtSPL的表达水平下调，而开花抑制基因AtSVP的表达则上调。结论 BoFLC3在植物开花过程中发挥关键作用，其异源过表达可显著延迟抽薹与开花，表明该基因在成花诱导途径中发挥负调控功能。

关键词: 青花菜, BoFLC3, 表达模式, 遗传转化, 开花

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

张静, 李圆圆, 王燕, 江雅洁, 杨寒冰, 柴文臣, 霍辰思, 罗丹, 阎世江. 青花菜BoFLC3的克隆与开花调控机制研究[J]. 生物技术通报, 2026, 42(9): 169-177.

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.

图/表 7

表1 过表达BoFLC3拟南芥T3代表型分析

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）

图1 BoFLC3的克隆及其编码氨基酸序列A：BoFLC3的PCR扩增（M：2 000 bp DNA marker；1：BoFLC3）；B：BoFLC3的核苷酸与氨基酸序列

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

图2 BoFLC3蛋白的序列比对与进化关系A：BoFLC3蛋白的氨基酸序列与其他物种FLC的比对；B：BoFLC3与其他物种FLC蛋白的系统发育树；红色框线代表本研究所用物种

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

图3 青花菜BoFLC3的组织表达模式CK：生长在25 ℃/17 ℃的对照植株；V15：17 ℃/9 ℃处理15 d的植株。*P<0.05；**P<0.01

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

图4 BoFLC3的亚细胞定位

Fig. 4 Subcellular localization of BoFLC3

图5 转基因拟南芥植株的鉴定A：转基因拟南芥中BoFLC3的PCR鉴定（M：2 000 bp DNA marker，1：pBI121-BoFLC3（阳性对照），2：野生型（阴性对照），3‒4：过表达BoFLC3拟南芥）；B：转基因株系中BoFLC3的相对表达量；C：过表达BoFLC3拟南芥植株表型。WT：野生型，OE：过表达BoFLC3植株。不同小写字母表示在P<0.05水平差异显著。下同

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

图6 过表达BoFLC3对拟南芥开花相关基因表达的影响

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

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