Bioinformatics Analysis and Flowering Regulation Function of FveBBX20 Gene in Woodland Strawberry

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

Abstract

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

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.

Figures/Tables 6

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

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

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

Fig. 3 Subcellular localization of FveBBX20 protein

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

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

References 33

[1]	Gangappa SN, Botto JF. The BBX family of plant transcription factors [J]. Trends Plant Sci, 2014, 19(7): 460-470.
[2]	刘嘉, 韩峰, 王雪源, 等. B-box家族蛋白在植物光信号转导中的作用机制进展 [J]. 植物生理学报, 2022, 58(6): 1023-1036.
	Liu J, Han F, Wang XY, et al. Recent advances on BBX protein family mediated light signal transduction in plants [J]. Plant Physiol J, 2022, 58(6): 1023-1036.
[3]	Khanna R, Kronmiller B, Maszle DR, et al. The Arabidopsis B-box zinc finger family [J]. Plant Cell, 2009, 21(11): 3416-3420.
[4]	Huang JY, Zhao XB, Weng XY, et al. The rice B-box zinc finger gene family: genomic identification, characterization, expression profiling and diurnal analysis [J]. PLoS One, 2012, 7(10): e48242.
[5]	Xu XH, Li WL, Yang SK, et al. Identification, evolution, expression and protein interaction analysis of genes encoding B-box zinc-finger proteins in maize [J]. J Integr Agric, 2023, 22(2): 371-388.
[6]	Chen SH, Jiang WQ, Yin JL, et al. Genome-wide mining of wheat B-BOX zinc finger (BBX) gene family provides new insights into light stress responses [J]. Crop Pasture Sci, 2021, 72(1): 17.
[7]	Feng Z, Li MY, Li Y, et al. Comprehensive identification and expression analysis of B-Box genes in cotton [J]. BMC Genomics, 2021, 22(1): 439.
[8]	Fan CM, Hu RB, Zhang XM, et al. Conserved CO-FT regulons contribute to the photoperiod flowering control in soybean [J]. BMC Plant Biol, 2014, 14: 9.
[9]	Bu X, Wang XJ, Yan JR, et al. Genome-wide characterization of B-box gene family and its roles in responses to light quality and cold stress in tomato [J]. Front Plant Sci, 2021, 12: 698525.
[10]	Wang YY, Zhai ZF, Sun YT, et al. Genome-wide identification of the B- BOX genes that respond to multiple ripening related signals in sweet cherry fruit [J]. Int J Mol Sci, 2021, 22(4): 1622.
[11]	Cao J, Yuan JL, Zhang YL, et al. Multi-layered roles of BBX proteins in plant growth and development [J]. Stress Biol, 2023, 3(1): 1.
[12]	Song ZQ, Bian YT, Liu JJ, et al. B-box proteins: Pivotal players in light-mediated development in plants [J]. J Integr Plant Biol, 2020, 62(9): 1293-1309.
[13]	Tripathi P, Carvallo M, Hamilton EE, et al. Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering [J]. Proc Natl Acad Sci USA, 2017, 114(1): 172-177.
[14]	Cheng H, Yu Y, Zhai YW, et al. An ethylene-responsive transcription factor and a B-box protein coordinate vegetative growth and photoperiodic flowering in Chrysanthemum [J]. Plant Cell Environ, 2023, 46(2): 440-450.
[15]	Wang Q, Wang LJ, Cheng H, et al. Two B-box proteins orchestrate vegetative and reproductive growth in summer Chrysanthemum [J]. Plant Cell Environ, 2024, 47(8): 2923-2935.
[16]	Ping Q, Cheng PL, Huang F, et al. The heterologous expression in Arabidopsis thaliana of a Chrysanthemum gene encoding the BBX family transcription factor CmBBX13 delays flowering [J]. Plant Physiol Biochem, 2019, 144: 480-487.
[17]	Yang YJ, Ma C, Xu YJ, et al. A zinc finger protein regulates flowering time and abiotic stress tolerance in Chrysanthemum by modulating gibberellin biosynthesis[J]. Plant Cell, 2014, 26(5): 2038-2054.
[18]	Tan JJ, Jin MN, Wang JC, et al. OsCOL10, a CONSTANS-like gene, functions as a flowering time repressor downstream of Ghd7 in rice [J]. Plant Cell Physiol, 2016, 57(4): 798-812.
[19]	Wu WX, Zheng XM, Chen DB, et al. OsCOL16, encoding a CONSTANS-like protein, represses flowering by up-regulating Ghd7 expression in rice [J]. Plant Sci, 2017, 260: 60-69.
[20]	Wu WX, Zhang YX, Zhang M, et al. The rice CONSTANS-like protein OsCOL15 suppresses flowering by promoting Ghd7 and repressing RID1 [J]. Biochem Biophys Res Commun, 2018, 495(1): 1349-1355.
[21]	邵妍丽, 卢贝, 贾思振, 等. 草莓FaWRKY70基因克隆与表达模式分析 [J]. 西北植物学报, 2024, 44(7): 1105-1112.
	Shao YL. Lu B, Jia SZ,et al. Cloning and expression analysis of the FaWRKY70 gene in strawberry [J]. Acta Botanica Boreali-Occidentalia Sinica, 2024, 44(7): 1105-1112.
[22]	张杨, 马宗桓, 毛娟, 等. 5种叶面肥对大棚草莓光合特性及品质的影响 [J]. 西北植物学报, 2024, 44(4): 562-571.
	Zhang Y, Ma ZH, Mao J, et al. Effects of five foliar fertilizers on photosynthetic characteristics and fruit quality of Fragaria ananassa in greenhouse [J]. Acta Botanica Boreali-Occidentalia Sinica, 2024, 44(4): 562-571.
[23]	叶云天, 娄可飞, 王熙然, 等. 森林草莓B-box基因家族全基因组序列鉴定与表达分析 [J] .分子植物育种, 2016, 14(4): 827-834.
	Ye YT, Lou KF, Wang XR, et al. Genome-wide identification and expression investigation of the B-box gene family in Fragaria vesca [J]. Molecular Plant Breeding, 2016, 14(4): 827-834.
[24]	Ye YT, Liu YQ, Li XL, et al. An evolutionary analysis of B-box transcription factors in strawberry reveals the role of FaBBx28c1 in the regulation of flowering time [J]. Int J Mol Sci, 2021, 22(21): 11766.
[25]	张奇瑞, 刘小林, 徐胜光, 等. 外源茉莉酸甲酯对连作草莓土壤养分和酶活性的影响 [J]. 西南农业学报, 2022, 35(8): 1764-1769.
	Zhang QR, Liu XL, Xu SG, et al. Effects of exogenous methyl jasmonate on soil nutrients and enzyme activities of continuous cropping strawberry [J]. Southwest China Journal of Agricultural Sciences, 2022, 35(8): 1764-1769.
[26]	He WZ, Liu H, Wu Z, et al. The AaBBX21-AaHY5 module mediates light-regulated artemisinin biosynthesis in Artemisia annua L [J]. J Integr Plant Biol, 2024, 66(8): 1735-1751.
[27]	邓巧灵, 华玮, 王华素, 等. 辣椒CcBBX20基因克隆和表达分析 [J]. 辣椒杂志, 2024, 22(1): 1-8.
	Deng QL, Hua W, Wang HS, et al. Cloning and expression analysis of gene CcBBX20 in pepper [J]. J China Capsicum, 2024, 22(1): 1-8.
[28]	周昕越, 蒋庆雪, 贾会丽, 等. 紫花苜蓿MsBBX20基因克隆及耐盐功能分析 [J]. 草业学报, 2024, 33(10): 55-73.
	Zhou XY, Jiang QX, Jia HL, et al. Cloning and salt-tolerance functional analysis of alfalfa MsBBX20 gene [J]. Acta Prataculturae Sinica, 2024, 33(10): 55-73.
[29]	陈新娜, 张利英, 喻锌, 等. 甘菊ClCOL16基因的克隆与功能初步研究 [J]. 西北植物学报, 2023, 43(3): 366-373.
	Chen XN, Zhang LY, Yu X, et al. Cloning and functional analysis of ClCOL16 from Chrysanthemum lavandulifolium [J]. Acta Bot Boreali Occidentalia Sin, 2023, 43(3): 366-373.
[30]	Kurokura T, Samad S, Koskela E, et al. Fragaria vesca CONSTANS controls photoperiodic flowering and vegetative development [J]. J Exp Bot, 2017, 68(17): 4839-4850.
[31]	Mouhu K, Kurokura T, Koskela EA, et al. The Fragaria vesca homolog of suppressor of overexpression of constans1 represses flowering and promotes vegetative growth [J]. Plant Cell, 2013, 25(9): 3296-3310.
[32]	Wang CQ, Sarmast MK, Jiang JS, et al. The transcriptional regulator BBX19 promotes hypocotyl growth by facilitating COP1-mediated EARLY FLOWERING3 degradation in Arabidopsis [J]. Plant Cell, 2015, 27(4): 1128-1139.
[33]	黄晓. 荔枝BBX基因响应UV-B和低温调控开花的机理研究 [D]. 广州: 华南农业大学, 2024.
	Huang X. BBX-mediated regulation of litchi flowering in response to UV-B and low temperature [D]. Guangzhou: South China Agricultural University, 2024.