森林草莓FveBBX20基因的生物信息学分析及开花调控功能

doi:10.13560/j.cnki.biotech.bull.1985.2025-0346

摘要/Abstract

摘要：

目的探究森林草莓FveBBX20基因在开花调控中的功能，为进一步探索其分子机制提供基础，也为草莓花期分子育种工作提供参考。方法以森林草莓‘Ruegen’为试材，通过RT-PCR方法对FveBBX20基因进行克隆，利用生物信息分析基因序列、蛋白质结构及进化关系，利用烟草瞬时表达观察蛋白定位情况，通过浸花法获得转基因拟南芥，观察转基因材料的开花表型，并利用实时荧光定量PCR分析组织表达和开花相关基因的表达情况。结果森林草莓FveBBX20的CDS序列全长为639 bp，编码212个氨基酸，蛋白质相对分子质量为23 958.62 Da，具有亲水性，含有2个B-box保守结构域，属于BBX家族第Ⅳ亚组；进化分析表明，FveBBX20与蔷薇科植物蕨麻和月季的同源基因亲缘关系较接近；亚细胞定位分析显示FveBBX20蛋白定位在细胞核，FveBBX20在森林草莓的不同组织中均有表达，在花中的表达量最高；在拟南芥中异源过表达FveBBX20基因，转基因株系表现出开花延迟的表型，同时开花相关基因AtCO、AtFT、AtSOC1和AtFUL的表达量均显著降低。结论森林草莓FveBBX20基因通过抑制开花相关基因的表达水平来延迟开花。

关键词: 草莓, BBX20, 基因克隆, 生物信息学分析, 亚细胞定位, 开花调控

Abstract:

Objective Exploring the function of FveBBX20 gene in flowering regulation provides foundation for further revealing the molecular mechanism, and a reference for molecular breeding of flowering in strawberry. Method Takingwoodland strawberry (Fragaria vesca 'Ruegen') as the experiment materials, the FveBBX20 gene was cloned by RT-PCR, and the gene sequences, protein structures and evolutionary relationships were analyzed by using bioinformatics websites and software. The protein localization was observed by tobacco transient expression. Transgenic Arabidopsisthaliana was obtained by flower dipping method, observing the flowering phenotype of transgenic materials. The expression of tissue and flowering-related genes was analyzed by real-time fluorescence quantitative PCR. Result The CDS length of FveBBX20 was 639 bp, encoding 212 amino acids and containing 2 B-box conserved domains. The CDS sequence of the woodland strawberry FveBBX20 was 639 bp in length, encoding 212 amino acids, and the relative molecular weight of protein was 23 958.62 Da, which was hydrophilic and contained two B-box conserved domains, belonging to the subgroup Ⅳ of the BBX family. Evolutionary analysis showed that FveBBX20 was closely related to its homologs from Argentina anserina and Rosa chinensis. Subcellular localization analysis showed that the FveBBX20 protein was localized in the nucleus, and FveBBX20 was expressed in different tissues of the woodland strawberry, with the highest expression level in flowers. Heterologous overexpression of the FveBBX20 gene in A. thaliana resulted in transgenic lines exhibiting delayed flowering phenotype, while the expressions of flowering-related genes AtCO, AtFT, AtSOC1, and AtFUL were significantly reduced. Conclusion FveBBX20 gene in the woodland strawberry delays flowering by inhibiting the expressions of flowering-related genes.

Key words: strawberry, BBX20, gene cloning, bioinformatics analysis, subcellular localization, flowering regulation

董向向, 缪百灵, 许贺娟, 陈娟娟, 李亮杰, 龚守富, 朱庆松. 森林草莓FveBBX20基因的生物信息学分析及开花调控功能[J]. 生物技术通报, 2025, 41(9): 115-123.

DONG Xiang-xiang, MIAO Bai-ling, XU He-juan, CHEN Juan-juan, LI Liang-jie, GONG Shou-fu, ZHU Qing-song. Bioinformatics Analysis and Flowering Regulation Function of FveBBX20 Gene in Woodland Strawberry[J]. Biotechnology Bulletin, 2025, 41(9): 115-123.

图/表 6

表1 引物信息

Table 1 Primer Information

基因名称 Name of gene	正向序列 Positive sequence （5′-3′）	反向序列 Reverse sequence （5′-3′）
FveBBX20	ATGAAGATCCGGTGCAATTTTT	TATGTAAGGTGCACTTTTAGCCTCA
AtACT2	AAGCTGGGGTTTTATGAATGG	TTGTCACACACAAGTGCATCAT
Fve26S	TAACCGCATCAGGTCTCCAA	CTCGAGCAGTTCTCCGACAG
AtCO	ATTCTGCAAACCCACTTGCT	CCTCCTTGGCATCCTTATCA
AtFT	CTGGAACAACCTTTGGCAAT	AGCCACTCTCCCTCTGACAA
AtFUL	GGAGAAGAAAACGGGTCAGC	ACTCGTTCGTAGTGGTAGGAC
AtSOC1	AATTCGCCAGCTCCAATATG	CCTCGATTGAGCATGTTCCTA

图1 FveBBX20基因克隆M. DL5000 Marker； 1. FveBBX20克隆片段

Fig. 1 Cloning of FveBBX20 geneM. DL5000 Marker; 1. Cloned fragments of FveBBX20

图2 FveBBX20蛋白的生物信息学分析A：FveBBX20基因的CDS和氨基酸序列；B：FveBBX20蛋白二级结构分析；C：FveBBX20蛋白三级结构分析；D：BBX20蛋白进化树分析；E：FveBBX20蛋白保守结构域；F：BBX20蛋白多序列比对

Fig. 2 Bioinformatics analysis of FveBBX20 proteinA: CDS and amino acid sequence of FveBBX20 gene. B: Analysis of secondary structure from FveBBX20 protein. C: Analysis of tertiary structure from FveBBX20 protein. E: Structural domain of FveBBX20 protein. F: Multiple sequence comparison of BBX20 proteins

图3 FveBBX20蛋白的亚细胞定位

Fig. 3 Subcellular localization of FveBBX20 protein

图4 不同组织FveBBX20相对表达量*表示在P<0.05水平差异显著， **表示在P<0.01水平差异显著。下同

Fig. 4 Relative expressions of FveBBX20 in different tissues* indicates statistically significant differences at P<0.05 level, and ** indicates statistically significant differences at P<0.01 level. The same below

图5 过表达FveBBX20拟南芥的鉴定和开花功能分析A：转基因拟南芥中FveBBX20的PCR鉴定， M：DL5000 marker， 1-3：转基因株系， 4：野生型Col-0； B：转基因拟南芥中FveBBX20基因表达量分析； C：转基因株系的开花表型； D：转基因株系的抽薹时间统计，不同小写字母表示不同株系间的显著性差异（P<0.05）； E-H：开花相关基因的表达量分析

Fig. 5 Identification and flowering function analysis of A. thaliana over-expressing FveBBX20 geneA: PCR identification of FveBBX20 in transgenic A. thaliana, M. DL5000 marker, 1-3. overexpression line, 4. wild typeCol-0. B: Expression analysis of FveBBX20 gene in transgenic A. thaliana. C: The flowering phenotype of transgenic lines. D: Statistics of bolting time of transgenic lines, different lowercase letters indicating significant differences between different strains (P<0.05). E-H: Expression level analysis of flowering-related genes

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