蔗茅EfBBX基因家族鉴定及冷胁迫表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0908

摘要/Abstract

摘要：

目的 B-box型锌指蛋白（BBX蛋白）在抵御植物非生物胁迫中具有重要作用。蔗茅具有良好的耐冷性，可为甘蔗的耐冷遗传改良提供基因资源。对蔗茅EfBBX基因家族成员鉴定和特征分析，并探究其在冷胁迫中的生物学功能。方法从蔗茅全基因组中鉴定出EfBBX基因家族成员，通过生物信息学方法分析其理化性质、系统进化、染色体定位、共线性分析、基因结构、保守结构域和启动子顺式元件；RT-qPCR试验对EfBBX基因家族成员进行冷胁迫下的表达模式分析。以蔗茅无性系E2为材料，克隆EfBBX4、EfBBX10和EfBBX18基因CDS序列，并将EfBBX18基因在本氏烟草中进行瞬时表达。结果在蔗茅全基因组中鉴定出19个EfBBX基因家族成员，划分为5个亚家族，其不均匀分布于蔗茅10条染色体中的6条；亚细胞定位预测显示EfBBX蛋白主要位于细胞核和叶绿体中；EfBBX基因家族的启动子区域含有大量激素、非生物胁迫响应以及光响应相关元件；RT-qPCR实验证实，10个基因可以被冷胁迫诱导表达；成功克隆了EfBBX4、EfBBX10和EfBBX18基因，其中蔗茅EfBBX18在本氏烟草中的瞬时表达可增强烟草的耐冷性。结论从蔗茅基因组中鉴定出19个EfBBX基因，10个基因均可以被冷胁迫诱导表达，成功克隆3个基因，其中EfBBX18在本氏烟草中的瞬时表达可增强烟草的耐冷性。

关键词: 蔗茅, BBX基因家族, 基因家族鉴定, 生物信息学, 冷胁迫, 表达模式, 瞬时表达, 耐冷性

Abstract:

Objective B-box zinc finger proteins (BBX proteins) play a crucial role in defending plants against abiotic stress. Erianthus fulvus has excellent cold tolerance and serves as a valuable genetic resource for improving cold resistance in sugarcane. We identified and characterized members of the EfBBX gene family in E. fulvus, and explored their biological functions under cold stress. Method Members of the EfBBX gene family were identified from the whole genome of E. fulvus. Bioinformatics methods were employed to analyze their physicochemical properties, phylogenetic relationships, chromosomal localization, synteny analysis, gene structure, conserved domains, and promoter cis-elements. RT-qPCR experiments were conducted to examine the expression patterns of EfBBX family members under cold stress. Using the E. fulvus clonal line E2 as material, the coding sequences (CDS) of EfBBX4, EfBBX10, and EfBBX18 were cloned. The EfBBX18 gene was then transiently expressed in Nicotiana benthamiana. Result Nineteen members of the EfBBX gene family were identified in the whole genome of E. fulvus, classified into five subfamilies and unevenly distributed across six of its ten chromosomes. Subcellular localization predictions indicated that EfBBX proteins were primarily localized in the nucleus and chloroplasts. The promoter regions of the EfBBX gene family contain numerous elements associated with hormone, abiotic stress response, and light response pathways. RT-qPCR experiments confirmed that ten genes were induced by cold stress. The EfBBX4, EfBBX10, and EfBBX18 genes were successfully cloned, and transient expression of E. fulvusEfBBX18 in Nicotiana benthamiana enhanced the plant’s cold tolerance. Conclusion Nineteen EfBBX genes are identified from the E. fulvus genome, with ten genes inducible by cold stress. Three of these genes are successfully cloned, and transient expression of EfBBX18 in N. benthamiana enhances the plant’s tolerance to cold.

Key words: Erianthusfulvus, BBX gene family, identification gene family, bioinformatics, cold stress, expression pattern, transient expression, cold resistance

农韦优, 赵昌祖, 钱禛锋, 丁倩, 王誉洁, 陈疏影, 何丽莲, 李富生. 蔗茅EfBBX基因家族鉴定及冷胁迫表达模式分析[J]. 生物技术通报, 2026, 42(2): 1-11.

NONG Wei-you, ZHAO Chang-zu, QIAN Zhen-feng, DING Qian, WANG Yu-jie, CHEN Shu-ying, HE Li-lian, LI Fu-sheng. Identification of the EfBBX Gene Family in Erianthus fulvus and Analysis of Its Expression Patterns Under Cold Stress[J]. Biotechnology Bulletin, 2026, 42(2): 1-11.

图/表 8

参考文献 52

[1]	高松, 王鹤潼. 低温胁迫下植物调控转录因子家族研究进展 [J]. 世界热带农业信息, 2025(6): 111-114.
	Gao S, Wang HT. Research progress of plant regulation transcription factor family under low temperature stress [J]. World Trop Agric Inf, 2025(6): 111-114.
[2]	郭佳强, 曾鑫, 陈孝平. 植物抗低温胁迫的研究进展 [J]. 武汉工程大学学报, 2023, 45(2): 119-125.
	Guojia Q, Zeng X, Chen XP. Advances in plant resistance to low temperature stress [J]. J Wuhan Inst Technol, 2023, 45(2): 119-125.
[3]	李子涵, 李建运, 王飞, 等. 植物对低温胁迫反应机制的研究进展 [J]. 农业与技术, 2024, 44(7): 134-138.
	Li ZH, Li JY, Wang F, et al. Research progress on response mechanism of plants to low temperature stress [J]. Agric Technol, 2024, 44(7): 134-138.
[4]	赵婷婷, 孙婷婷, 王俊刚, 等. 甘蔗育种研究进展 [J]. 中国科学: 生命科学, 2024, 54(10): 1814-1832.
	Zhao TT, Sun TT, Wang JG, et al. The research progress on sugarcane breeding [J]. Sci Sin Vitae, 2024, 54(10): 1814-1832.
[5]	周慧文, 陆桂军, 吴建明, 等. 中国甘蔗种业发展研究进展 [J]. 广西科学, 2023, 30(3): 421-433.
	Zhou HW, Lu GJ, Wu JM, et al. Research progress of sugarcane seed industry in China [J]. Guangxi Sci, 2023, 30(3): 421-433.
[6]	胡水凤. 甘蔗抗逆转基因研究进展 [J]. 广西糖业, 2022, 42(5): 18-21.
	Hu SF. Research progress of sugarcane anti-reversion genes [J]. Guangxi Sugar Ind, 2022, 42(5): 18-21.
[7]	张志勇, 雷朝云, 蒙秋伊, 等. 甘蔗抗逆基因工程育种研究进展 [J]. 核农学报, 2012, 26(3): 471-477.
	Zhang ZY, Lei CY, Meng QY, et al. Research progress on genetic engineering of stress tolerance in sugarcane [J]. J Nucl Agric Sci, 2012, 26(3): 471-477.
[8]	萧凤回, 李富生, 何丽莲, 等. 甘蔗近缘野生种蔗茅（Erianthus rufipilus）的研究 [J]. 甘蔗, 1996(2): 1-6.
	Xiao FH, Li FS, He LL, et al. Study on the wild species of sugarcane (Erianthus rufipilus) [J]. Subtrop Agric Res, 1996(2): 1-6.
[9]	李富生, 林位夫, 何顺长. 开发利用蔗茅野生种质资源的思考 [J]. 资源开发与市场, 2004, 20(4): 266-270.
	Li FS, Lin WF, He SC. Suggestions on developing and utilization of Erianthus fulvus wild species [J]. Resour Dev Mark, 2004, 20(4): 266-270.
[10]	王丽萍, 蔡青, 陆鑫, 等. 甘蔗近缘属野生种滇蔗茅（Erianthus rockii）的种质创新利用 [J]. 中国糖料, 2008, 30(2): 8-11.
	Wang LP, Cai Q, Lu X, et al. Study of wild species Erianthus rockii germplasm innovation and use [J]. Sugar Crops China, 2008, 30(2): 8-11.
[11]	王秋松. 甘蔗与滇蔗茅后代染色体遗传及利用价值研究 [D]. 福州: 福建农林大学, 2022.
	Wang QS. Research on chromosome inheritance and application value of the progenies of Saccharum officinarum and Erianthus rockii [D]. Fuzhou: Fujian Agriculture and Forestry University, 2022.
[12]	钱禛锋, 赵昌祖, 万慧兰, 等. 甘蔗近缘野生种蔗茅的研究进展和利用潜力 [J]. 植物遗传资源学报, 2025, 26(8): 1485-1498.
	Qian ZF, Zhao CZ, Wan HL, et al. Research progress and utilization potential of wild sugarcane species Erianthus fulvus ness [J]. J Plant Genet Resour, 2025, 26(8): 1485-1498.
[13]	杨宁, 从青, 程龙军. 植物BBX转录因子基因家族的研究进展 [J]. 生物工程学报, 2020, 36(4): 666-677.
	Yang N, Cong Q, Cheng LJ. BBX transcriptional factors family in plants-a review [J]. Chin J Biotechnol, 2020, 36(4): 666-677.
[14]	Gangappa SN, Botto JF. The BBX family of plant transcription factors [J]. Trends Plant Sci, 2014, 19(7): 460-470.
[15]	Gao LL, Xu S, Zhang JM, et al. Promotion of seedling germination in Arabidopsis by B-box zinc-finger protein BBX32 [J]. Curr Biol, 2024, 34(14): 3152-3164.e6.
[16]	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.
[17]	Cao J, Liang YX, Yan TT, et al. The photomorphogenic repressors BBX28 and BBX29 integrate light and brassinosteroid signaling to inhibit seedling development in Arabidopsis [J]. Plant Cell, 2022, 34(6): 2266-2285.
[18]	Bian YT, Song ZL, Liu CS, et al. The BBX7/8-CCA1/LHY transcription factor cascade promotes shade avoidance by activating PIF4 [J]. New Phytol, 2025, 245(2): 637-652.
[19]	Song ZQ, Yan TT, Liu JJ, et al. BBX28/BBX29, HY5 and BBX30/31 form a feedback loop to fine-tune photomorphogenic development [J]. Plant J, 2020, 104(2): 377-390.
[20]	Yang CL, Zhang SW, Zou QJ, et al. CiBBX19 negatively regulates the flowering time of Chrysanthemum indicum by recruiting CiBBX5 to inhibit the expression of CiFTL3 [J]. Plant Physiol Biochem, 2025, 227: 110115.
[21]	Fu JL, Liao L, Jin JJ, et al. A transcriptional cascade involving BBX22 and HY5 finely regulates both plant height and fruit pigmentation in Citrus [J]. J Integr Plant Biol, 2024, 66(8): 1752-1768.
[22]	邓军, 罗正良, 朱菲莹, 等. 水稻B-box锌指蛋白基因OsBBX26的克隆及表达分析 [J]. 南方农业学报, 2020, 51(7): 1606-1613.
	Deng J, Luo ZL, Zhu FY, et al. Cloning and expression analysis of B-box zinc-finger protein gene OsBBX26 in rice [J]. J South Agric, 2020, 51(7): 1606-1613.
[23]	齐小坤, 孟祥熹, 赵志明, 等. 毛果杨BBX基因家族鉴定与生物信息学分析 [J]. 河北农业大学学报, 2023, 46(5): 41-49.
	Qi XK, Meng XX, Zhao ZM, et al. Identification and bioinformatics analysis of BBX gene family in Populus trichocarpa [J]. J Hebei Agric Univ, 2023, 46(5): 41-49.
[24]	宁露云, 李蓓, 李佩, 等. 矮牵牛B-box型锌指蛋白基因PhBBX8的克隆与表达分析 [J]. 园艺学报, 2014, 41(12): 2437-2445.
	Ning LY, Li B, Li P, et al. Cloning and expression analysis of a B-box-type zinc-finger protein gene PhBBX8 in Petunia hybrida [J]. Acta Hortic Sin, 2014, 41(12): 2437-2445.
[25]	Li YP, Shi YT, Li MZ, et al. The CRY2-COP1-HY5-BBX7/8 module regulates blue light-dependent cold acclimation in Arabidopsis [J]. Plant Cell, 2021, 33(11): 3555-3573.
[26]	An JP, Wang XF, Zhang XW, et al. Apple B-box protein BBX37 regulates jasmonic acid mediated cold tolerance through the JAZ-BBX37-ICE1-CBF pathway and undergoes MIEL1-mediated ubiquitination and degradation [J]. New Phytol, 2021, 229(5): 2707-2729.
[27]	Zhang H, Chen MJ, Luo XB, et al. Overexpression of StBBX14 enhances cold tolerance in potato [J]. Plants, 2024, 14(1): 18.
[28]	Song JN, Lin R, Tang MJ, et al. SlMPK1- and SlMPK2-mediated SlBBX17 phosphorylation positively regulates CBF-dependent cold tolerance in tomato [J]. New Phytol, 2023, 239(5): 1887-1902.
[29]	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.
[30]	Zhu YF, Zhu GT, Xu R, et al. A natural promoter variation of SlBBX31 confers enhanced cold tolerance during tomato domestication [J]. Plant Biotechnol J, 2023, 21(5): 1033-1043.
[31]	Wang S, Shen YR, Deng DY, et al. Orthogroup and phylotranscriptomic analyses identify transcription factors involved in the plant cold response: a case study of Arabidopsis BBX29 [J]. Plant Commun, 2023, 4(6): 100684.
[32]	Lyu GZ, Li DB, Li SS. Bioinformatics analysis of BBX family genes and its response to UV-B in Arabidopsis thaliana [J]. Plant Signal Behav, 2020, 15(9): 1782647.
[33]	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.
[34]	Shalmani A, Jing XQ, Shi Y, et al. Characterization of B-BOX gene family and their expression profiles under hormonal, abiotic and metal stresses in Poaceae plants [J]. BMC Genomics, 2019, 20(1): 27.
[35]	Chu ZN, Wang X, Li Y, et al. Genomic organization, phylogenetic and expression analysis of the B-BOX gene family in tomato [J]. Front Plant Sci, 2016, 7: 1552.
[36]	Yin LL, Wu RG, An RL, et al. Genome-wide identification, molecular evolution and expression analysis of the B-box gene family in mung bean (Vigna radiata L.) [J]. BMC Plant Biol, 2024, 24(1): 532.
[37]	Kui L, Majeed A, Wang XH, et al. A chromosome-level genome assembly for Erianthus fulvus provides insights into its biofuel potential and facilitates breeding for improvement of sugarcane [J]. Plant Commun, 2023, 4(4): 100562.
[38]	Singh S, Chhapekar SS, Ma YB, et al. Genome-wide identification, evolution, and comparative analysis of B-Box genes in Brassica rapa, B. oleracea, and B. napus and their expression profiling in B. Rapa in response to multiple hormones and abiotic stresses [J]. Int J Mol Sci, 2021, 22(19): 10367.
[39]	Liu Y, Wang YX, Liao J, et al. Identification and characterization of the BBX gene family in Bambusa pervariabilis × Dendrocalamopsis grandis and their potential role under adverse environmental stresses [J]. Int J Mol Sci, 2023, 24(17): 13465.
[40]	马文婧, 刘震, 李志涛, 等. 马铃薯BBX基因家族的全基因组鉴定及表达分析 [J]. 作物学报, 2022(11): 2797-2812.
	Ma WJ, Liu Z, Li ZT, et al. Whole genome identification and expression analysis of potato BBX gene family [J]. Acta Agronomica Sinica, 2022(11): 2797-2812.
[41]	殷丽丽, 邢宝龙, 陈晓亮, 等. 大豆BBX基因家族的鉴定及表达 [J]. 西北农林科技大学学报: 自然科学版, 2022, 50(6): 35-45.
	Yin LL, Xing BL, Chen XL, et al. Genome-wide identification and expression patterns of BBX gene family members in Glycine max [J]. J Northwest A F Univ Nat Sci Ed, 2022, 50(6): 35-45.
[42]	Liu X, Li R, Dai YQ, et al. Genome-wide identification and expression analysis of the B-box gene family in the apple (Malus domestica Borkh.) genome [J]. Mol Genet Genomics, 2018, 293(2): 303-315.
[43]	翟海涛, 余月, 陈蕾, 等. 芥菜BjuBBX基因家族全基因组鉴定与分析 [J]. 江西农业学报, 2023, 35(11): 40-50.
	Zhai HT, Yu Y, Chen L, et al. Genome identification and analysis of BjuBBX gene family in mustard [J]. Acta Agric Jiangxi, 2023, 35(11): 40-50.
[44]	Wu ZL, Fu DW, Gao XN, et al. Characterization and expression profiles of the B-box gene family during plant growth and under low-nitrogen stress in Saccharum [J]. BMC Genomics, 2023, 24(1): 79.
[45]	Cheng XR, Lei SY, Li J, et al. In silico analysis of the wheat BBX gene family and identification of candidate genes for seed dormancy and germination [J]. BMC Plant Biol, 2024, 24(1): 334.
[46]	杨倩, 苏旭, 刘玉萍, 等. 沙鞭SnRK2基因家族鉴定及干旱胁迫下表达分析 [J]. 草地学报, 2025, 33(10): 3173-3184.
	Yang Q, Su X, Liu YP, et al. Identification of SnRK2 gene family in Psammochloa villosa and analysis of their expression under drought stress [J]. Acta Agrestia Sin, 2025, 33(10): 3173-3184.
[47]	Chandra Malakar B, Singh S, Garhwal V, et al. BBX24 and BBX25 antagonize the function of thermosensor ELF3 to promote PIF4-mediated thermomorphogenesis in Arabidopsis [J]. Plant Commun, 2025, 6(7): 101391.
[48]	Gao XR, Zhang H, Li X, et al. The B-box transcription factor IbBBX29 regulates leaf development and flavonoid biosynthesis in sweet potato [J]. Plant Physiol, 2023, 191(1): 496-514.
[49]	Feng XJ, Li JF, Tang ZR, et al. BjuBBX6-1 interacts with BjuNF-YB2/3 to regulate flowering time and drought tolerance in Brassica juncea [J]. Hortic Plant J, 2025
[50]	Bandara WW, Wijesundera WSS, Hettiarachchi C. Rice and Arabidopsis BBX proteins: toward genetic engineering of abiotic stress resistant crops [J]. 3 Biotech, 2022, 12(8): 164.
[51]	Dai YQ, Lu Y, Zhou Z, et al. B-box containing protein 1 from Malus domestica (MdBBX1) is involved in the abiotic stress response [J]. PeerJ, 2022, 10: e12852.
[52]	Qian ZF, He LL, Li FS. Understanding cold stress response mechanisms in plants: an overview [J]. Front Plant Sci, 2024, 15: 1443317.

基因 Gene	基因IDGene ID	氨基酸数量 Number of amino Acids （aa）	相对分子量 Molecular Weight （Da）	等电点PI	不稳定指数 Instability index	脂肪指数 Aliphatic index	亚细胞定位 Subcellular localization
EfBBX1	Erufi.01G0010670.t1	420	45 969.84	5.88	55.68	64.21	叶绿体 Chloroplast
EfBBX2	Erufi.03G0003170.t1	358	37 106.31	5.42	51.18	65.67	细胞核 Nuclear
EfBBX3	Erufi.04G0006700.t1	373	38 846.33	5.80	45.40	65.39	叶绿体 Chloroplast
EfBBX4	Erufi.04G0019680.t1	257	27 606.01	4.95	43.19	76.54	细胞质 Cytoplasmic
EfBBX5	Erufi.04G0020000.t1	328	34 599.45	5.12	44.90	66.10	细胞质 Cytoplasmic
EfBBX6	Erufi.04G0022180.t1	276	28 816.93	5.38	61.93	63.91	细胞核 Nuclear
EfBBX7	Erufi.04G0026320.t1	405	44 398.46	5.30	50.12	61.73	细胞核 Nuclear
EfBBX8	Erufi.04G0026680.t1	374	41 614.82	6.42	56.63	70.16	叶绿体 Chloroplast
EfBBX9	Erufi.04G0026930.t1	476	51 626.87	5.68	65.17	63.87	叶绿体 Chloroplast
EfBBX10	Erufi.06G0013060.t1	255	27 181.54	4.80	49.61	69.76	细胞核 Nuclear
EfBBX11	Erufi.06G0013390.t1	354	37 454.85	5.46	38.07	63.73	叶绿体 Chloroplast
EfBBX12	Erufi.06G0016230.t1	261	28 309.31	4.89	70.95	61.07	细胞核 Nuclear
EfBBX13	Erufi.09G0008130.t1	327	34 135.01	5.07	56.04	63.15	细胞核 Nuclear
EfBBX14	Erufi.10G0004850.t1	481	52 067.46	6.60	61.19	67.53	叶绿体 Chloroplast
EfBBX15	Erufi.10G0011520.t1	459	49 201.79	5.42	46.14	68.82	细胞质 Cytoplasmic
EfBBX16	Erufi.10G0012110.t1	403	43 490.53	5.31	48.03	69.58	细胞质 Cytoplasmic
EfBBX17	Erufi.10G0012650.t1	404	43 489.41	5.30	46.75	65.50	细胞核 Nuclear
EfBBX18	Erufi.10G0020560.t1	366	39 089.78	5.76	43.58	69.48	细胞质 Cytoplasmic
EfBBX19	Erufi.10G0025500.t1	303	31 346.08	4.83	57.21	70.99	叶绿体 Chloroplast

基因 Gene	基因IDGene ID	氨基酸数量 Number of amino Acids （aa）	相对分子量 Molecular Weight （Da）	等电点PI	不稳定指数 Instability index	脂肪指数 Aliphatic index	亚细胞定位 Subcellular localization
EfBBX1	Erufi.01G0010670.t1	420	45 969.84	5.88	55.68	64.21	叶绿体 Chloroplast
EfBBX2	Erufi.03G0003170.t1	358	37 106.31	5.42	51.18	65.67	细胞核 Nuclear
EfBBX3	Erufi.04G0006700.t1	373	38 846.33	5.80	45.40	65.39	叶绿体 Chloroplast
EfBBX4	Erufi.04G0019680.t1	257	27 606.01	4.95	43.19	76.54	细胞质 Cytoplasmic
EfBBX5	Erufi.04G0020000.t1	328	34 599.45	5.12	44.90	66.10	细胞质 Cytoplasmic
EfBBX6	Erufi.04G0022180.t1	276	28 816.93	5.38	61.93	63.91	细胞核 Nuclear
EfBBX7	Erufi.04G0026320.t1	405	44 398.46	5.30	50.12	61.73	细胞核 Nuclear
EfBBX8	Erufi.04G0026680.t1	374	41 614.82	6.42	56.63	70.16	叶绿体 Chloroplast
EfBBX9	Erufi.04G0026930.t1	476	51 626.87	5.68	65.17	63.87	叶绿体 Chloroplast
EfBBX10	Erufi.06G0013060.t1	255	27 181.54	4.80	49.61	69.76	细胞核 Nuclear
EfBBX11	Erufi.06G0013390.t1	354	37 454.85	5.46	38.07	63.73	叶绿体 Chloroplast
EfBBX12	Erufi.06G0016230.t1	261	28 309.31	4.89	70.95	61.07	细胞核 Nuclear
EfBBX13	Erufi.09G0008130.t1	327	34 135.01	5.07	56.04	63.15	细胞核 Nuclear
EfBBX14	Erufi.10G0004850.t1	481	52 067.46	6.60	61.19	67.53	叶绿体 Chloroplast
EfBBX15	Erufi.10G0011520.t1	459	49 201.79	5.42	46.14	68.82	细胞质 Cytoplasmic
EfBBX16	Erufi.10G0012110.t1	403	43 490.53	5.31	48.03	69.58	细胞质 Cytoplasmic
EfBBX17	Erufi.10G0012650.t1	404	43 489.41	5.30	46.75	65.50	细胞核 Nuclear
EfBBX18	Erufi.10G0020560.t1	366	39 089.78	5.76	43.58	69.48	细胞质 Cytoplasmic
EfBBX19	Erufi.10G0025500.t1	303	31 346.08	4.83	57.21	70.99	叶绿体 Chloroplast