旱柳SmERF B2-8基因在响应淹水胁迫中的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1134

摘要/Abstract

摘要：

目的阐明旱柳中SmERF B2-8基因在响应淹水胁迫中的生物学功能，验证其是否为调控耐淹性的关键因子。方法克隆旱柳中的 SmERF B2-8基因，并进行相关生物信息学分析，通过RT-qPCR揭示SmERF B2-8基因在不同品种柳树淹水胁迫不同时期表达量的变化。进一步构建SmERF B2-8基因的过表达载体获得转基因拟南芥，并开展拟南芥的缺氧胁迫响应研究；同时构建基因沉默载体进行旱柳的VIGS实验，系统验证该基因在淹水胁迫中的生物学功能。结果旱柳中SmERF B2-8基因的编码区（CDS）序列全长为948 bp，启动子区域分析发现其中含有与缺氧相关的顺式作用元件。RT-qPCR结果显示SmERF B2-8基因的表达量随淹水时间延长而上调，相较于0 h和4 h，在淹水12 h基因的表达量出现显著上调。缺氧胁迫实验发现相对于野生型拟南芥，SmERF B2-8转基因拟南芥对缺氧胁迫的耐受性增强。VIGS实验结果显示，在旱柳中敲低SmERF B2-8基因后，相较于对照组，其对淹水胁迫的耐受性显著降低。结论旱柳SmERF B2-8基因启动子含缺氧响应顺式元件，该基因表达量在淹水胁迫后上调；过表达SmERF B2-8基因拟南芥缺氧耐受性增强，而旱柳中该基因沉默后耐淹性下降。旱柳SmERF B2-8基因是响应淹水胁迫的正调控因子。

关键词: 旱柳, ERF转录因子, 载体构建, 缺氧胁迫, VIGS实验

Abstract:

Objective This study aimed to elucidate the biological function of the SmERF B2-8 gene in Salix matsudana Koidz. in response to waterlogging stress and verify whether it is a key factor regulating the tolerance to waterlogging. Method The SmERF B2-8 gene in S. matsudana Koidz. was cloned and analyzed bioinformatically. RT-qPCR was performed to measure its expression in different Salix varieties under waterlogging at various time. The overexpressing vector of SmERF B2-8 was further constructed and transgenic Arabidopsis thaliana was obtained. The study on the hypoxic stress response of A. thaliana was conducted. Concurrently, a gene silencing vector was constructed to conduct VIGS experiments on S. matsudana Koidz. andthe biological function of the gene under waterlogging stress was systematically verified. Result The coding sequence (CDS) of the SmERF B2-8 gene in S. matsudana Koidz. is 948 bp in length. Analysis of the promoter region of SmERF B2-8 revealed the presence of oxygen-related cis-acting elements. RT-qPCR results showed that the expression of the SmERF B2-8 gene increased with prolonged waterlogging time, with significantly upregulated expression at 12 h compared to 0 and 4 h. Transgenic Arabidopsis experiments showed that, SmERF B2-8-overexpressing transgenic Arabidopsis exhibited better tolerance to hypoxia stress, compared to wild-type Arabidopsis. VIGS experimental results demonstrated that when the SmERF B2-8 gene was silenced in S. matsudana Koidz., the tolerance to waterlogging stress was significantly reduced compared to the control group. Conclusion The SmERF B2-8 gene promoter in S. matsudana Koidz. contains hypoxia-responsive cis-acting elements, and its gene expression is upregulated after waterlogging stress. SmERF B2-8 transgenic A. thaliana exhibits enhanced hypoxia tolerance, while silencing of this gene in S. matsudana Koidz. reduced its tolerance to waterlogging. SmERF B2-8 gene in S. matsudana Koidz. is a positive regulatory factor in response to waterlogging stress.

Key words: Salix matsudana Koidz., ERF transcription factor, vector construction, hypoxia stress, VIGS experiment

李梦茹, 钱超楠, 阚思蔚, 刘国元, 张健, 陈艳红. 旱柳SmERF B2-8基因在响应淹水胁迫中的功能研究[J]. 生物技术通报, 2026, 42(7): 204-213.

LI Meng-ru, QIAN Chao-nan, KAN Si-wei, LIU Guo-yuan, ZHANG Jian, CHEN Yan-hong. Functional Study of Salix matsudanaSmERF B2-8 Gene in Response to Waterlogging Stress[J]. Biotechnology Bulletin, 2026, 42(7): 204-213.

图/表 7

表1 本研究所用引物

Table 1 Primers used in the study

引物名称 Primer name	上游引物 Forward sequence （5'-3'）	下游引物 Reverse sequence （5'-3'）
SmERF B2-8	TCGTTTCCTCGCACTACA	ACAAAACCACCGGGAAAA
pYL156-SmERF B2-8	TAAGGTTACCGAATTCC TGAGGAGTTAATCGCGT	CGAGACGCGTGAGCTCA CAAAACCACCGGGAAAA
pWM101-SmERF B2-8	TCGAGCTTTCGCGAGCTC GGTACCATGTGTGGCGGT	CTCACCATGGTGGCGACC GGTACCTTACAGAGGTGC
qPCR-SmERF B2-8	ATGTGTGGCGGTGCTATTTT	GGGCTCAGAAAGGAATCAAA
pYL156-SmPDS	AATCAATGGGCCATGCCCTG	GCGGAGAAGAGCGAAAGGAT

图1 SmERF B2-8基因的PCR扩增及载体构建A：PCR扩增产物；B：过表达载体；C：沉默载体

Fig. 1 PCR amplification and vector construction of the SmERF B2-8 geneA: PCR amplification products. B. Overexpression vectors. C. Silencing vectors

图2 SmERF B2-8蛋白理化性质分析及蛋白质结构预测结果A：二级结构；B：三级结构；C：保守结构域；D：跨膜结构域；E：亲/疏水性

Fig. 2 Physicochemical properties analysis and protein structure predicting results of SmERF B2-8A: Secondary structure. B: Tertiary structure. C: Conserved structural domains. D: Transmembrane domains. E: Hydrophobicity/hydrophilicity

图3 SmERF B2-8的系统进化与启动子顺式作用元件分析A：系统发育树；B：SmERF B2-8蛋白同源序列比对；C：SmERF B2-8启动子顺式作用元件分析

Fig. 3 Phylogenetic and promoter cis-acting element analysis of SmERF B2-8A: Phylogenetic tree. B: SmERF B2-8 protein homologous sequence alignment. C: SmERF B2-8 promoter cis-acting element analysis

图4 不同品种柳树SmERF B2-8基因在淹水胁迫下的相对表达量*P<0.05，**P<0.01，***P<0.001，下同

Fig. 4 Relative expressions of the SmERF B2-8 gene in different willows varieties under submergence stress

图5 WT和SmERF B2-8转基因拟南芥表型指标和生理指标的比较A：正常氧和低氧条件下，WT和SmERF B2-8转基因拟南芥的表型比较；B：正常氧条件下的根长；C：正常氧条件下的鲜重；D：低氧条件下的根长；E：低氧条件下的鲜重；F：测定的MDA的含量

Fig. 5 Comparison of phenotypic and physiological indicators between wild type (WT) and transgenic Arabidopsis overexpressing SmERF B2-8A: Phenotypic comparison between WT and SmERF B2-8 transgenic Arabidopsis under normoxic and hypoxic conditions. B: Root length under normal conditions. C: Fresh weight under normal conditions. D: Root length under hypoxic conditions. E: Fresh weight under hypoxic conditions. F: Measured MDA content

图6 SmERF B2-8沉默降低柳树对淹水胁迫的耐受性A：淹水前后表型差异；B：转基因植株SmERF B2-8的相对表达量；C：根鲜重；D：根干重；E：MDA含量

Fig. 6 Silencing of SmERF B2-8 reduces the tolerance of willow to flood stressA: Phenotypic differences before and after submergence. B: Relative expression levels of the transgenic plant SmERF B2-8. C: Fresh weight of the roots. D: Dry weight of the roots. E: content MDA

参考文献 38

[1]	胡耀芳, 李奥, 殷佳慧, 等. 基于转录组的ABA介导的旱柳枝条失水响应 [J]. 北京林业大学学报, 2025, 47(3): 19-27.
	Hu YF, Li A, Yin JH, et al. Transcriptome based ABA mediated dehydration response of Salix matsudana branches [J]. J Beijing For Univ, 2025, 47(3): 19-27.
[2]	方琴. 水杉幼苗对淹水和干旱胁迫的生理响应 [D]. 上海: 华东师范大学, 2021.
	Fang Q. Physiological responses of Metasequoia glyptostroboides seedlings to flooding and drought stress [D]. Shanghai: East China Normal University, 2021.
[3]	Zhang YS, Chen XJ, Geng SY, et al. A review of soil waterlogging impacts, mechanisms, and adaptive strategies [J]. Front Plant Sci, 2025, 16: 1545912.
[4]	Renziehausen T, Chaudhury R, Hartman S, et al. A mechanistic integration of hypoxia signaling with energy, redox, and hormonal cues [J]. Plant Physiol, 2024, 197(1): kiae596.
[5]	朱婧, 杨城, 刘思, 等. 淹水胁迫下6种北方花灌木光合特性及耐涝性评价 [J]. 植物生理学报, 2024, 60(4): 725-738.
	Zhu J, Yang C, Liu S, et al. Photosynthetic characteristics and waterlogging tolerance evaluation of six northern flowering shrubs under waterlogging stress [J]. Plant Physiol J, 2024, 60(4): 725-738.
[6]	邵文靖, 敖特根白音, 郎明林. AP2/ERF转录因子对植物非生物胁迫的应答机制研究进展 [J]. 分子植物育种, 2020, 18(15): 4981-4988.
	Shao WJ, Ao T, Lang ML. Research advances on the mechanism of AP2/ERF transcriptional factors in response to abiotic stresses in plants [J]. Mol Plant Breed, 2020, 18(15): 4981-4988.
[7]	黄倩慧, 田博雯, 华炫, 等. AP2/ERF转录因子家族响应木本植物非生物胁迫的研究进展 [J]. 南通大学学报: 自然科学版, 2025, 24(1): 74-84.
	Huang QH, Tian BW, Hua X, et al. Research progress of AP2/ERF transcription factor family in response to abiotic stress in woody plants [J]. J Nantong Univ Nat Sci Ed, 2025, 24(1): 74-84.
[8]	张麒, 陈静, 李俐, 等. 植物AP2/ERF转录因子家族的研究进展 [J]. 生物技术通报, 2018, 34(8): 1-7.
	Zhang Q, Chen J, Li L, et al. Research progress on plant AP2/ERF transcription factor family [J]. Biotechnol Bull, 2018, 34(8): 1-7.
[9]	Zhang J, Shi SZ, Jiang YN, et al. Genome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow (Salix matsudana) [J]. PeerJ. 2021, 9: e11076.
[10]	邵文靖, 石洁, 张普, 等. ERF转录因子调控生物胁迫反应的研究进展 [J]. 生物技术通报, 2021, 37(3): 136-143.
	Shao WJ, Shi J, Zhang P, et al. Research progress of ERF transcription factors in regulating biological stress responses [J]. Biotechnol Bull, 2021, 37(3): 136-143.
[11]	Yu F, Liang K, Fang T, et al. A group VII ethylene response factor gene, ZmEREB180, coordinates waterlogging tolerance in maize seedlings [J]. Plant Biotechnol J, 2019, 17(12): 2286-2298.
[12]	Rong W, Qi L, Wang AY, et al. The ERF transcription factor TaERF3 promotes tolerance to salt and drought stresses in wheat [J]. Plant Biotechnol J, 2014, 12(4): 468-479.
[13]	Chen YH, Dai YH, Li YX, et al. Overexpression of the Salix matsudana SmAP2-17 gene improves Arabidopsis salinity tolerance by enhancing the expression of SOS3 and ABI5 [J]. BMC Plant Biol, 2022, 22: 102.
[14]	Zhu YQ, Liu Y, Zhou KM, et al. Overexpression of ZmEREBP60 enhances drought tolerance in maize [J]. J Plant Physiol, 2022, 275: 153763.
[15]	Zang ZY, Lv Y, Liu S, et al. A novel ERF transcription factor, ZmERF105, positively regulates maize resistance to Exserohilum turcicum [J]. Front Plant Sci, 2020, 11: 850.
[16]	Zhu YX, Zhang XM, Zhang QH, et al. The transcription factors VaERF16 and VaMYB306 interact to enhance resistance of grapevine to Botrytis cinerea infection [J]. Mol Plant Pathol, 2022, 23(10): 1415-1432.
[17]	王雅慧, 李彤, 黄莹, 等. 番茄2个ERF-B1亚族转录因子基因的克隆及其对生物和非生物胁迫响应 [J]. 核农学报, 2019, 33(10): 1893-1904.
	Wang YH, Li T, Huang Y, et al. Cloning and expression analysis under biotic and abiotic stresses of two ERF-B1 group transcription factor genes from Solanum lycopersicum [J]. J Nucl Agric Sci, 2019, 33(10): 1893-1904.
[18]	Li CW, Wang LK, Su JS, et al. A group VIIIa ethylene-responsive factor, CmERF4, negatively regulates waterlogging tolerance in Chrysanthemum [J]. J Exp Bot, 2024, 75(5): 1479-1492.
[19]	Liu DF, Chen XJ, Liu JQ, et al. The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance [J]. J Exp Bot, 2012, 63(10): 3899-3911.
[20]	Chen M, Liu X, Jiang SH, et al. Transcriptomic and functional analyses reveal that PpGLK1 regulates chloroplast development in peach (Prunus persica) [J]. Front Plant Sci, 2018, 9: 34.
[21]	钱超楠, 徐孙然, 李梦茹, 等. 旱柳NAC基因家族的全基因组鉴定及其在淹水和盐胁迫下的表达分析 [J]. 南方农业学报, 2024, 55(10): 3056-3070.
	Qian CN, Xu SR, Li MR, et al. Whole genomic identification of NAC gene family and expres-Sion analysis under flooding stress and salt stress in Salix matsudana [J]. J South Agric, 2024, 55(10): 3056-3070.
[22]	Chen YH, Deng MC, Huang QH, et al. SmDREB A1-10 is required for SmTTF30-mediated hypoxia stress tolerance in Salix matsudana [J]. Plant Cell Environ, 2025, 48(6): 4415-4429.
[23]	Kong LF, Song Q, Wei HB, et al. The AP2/ERF transcription factor PtoERF15 confers drought tolerance via JA-mediated signaling in Populus [J]. New Phytol, 2023, 240(5): 1848-1867.
[24]	聂功平, 陈敏敏, 杨柳燕, 等. 植物响应淹水胁迫的研究进展 [J]. 中国农学通报, 2021, 37(18): 57-64.
	Nie GP, Chen MM, Yang LY, et al. Plant response to waterlogging stress: research progress [J]. Chin Agric Sci Bull, 2021, 37(18): 57-64.
[25]	Cui MY, Du ZX, Li XY, et al. Physiological and ecological characteristics and reproductive responses of Phragmites australis to dry-wet conditions in inland saline marshes of Northeast China [J]. PeerJ, 2022, 10: e14269.
[26]	Feng K, Wang X, Zhou Q, et al. Waterlogging priming enhances hypoxia stress tolerance of wheat offspring plants by regulating root phenotypic and physiological adaption [J]. Plants, 2022, 11(15): 1969.
[27]	Maric A, Hartman S. Ethylene controls translational gatekeeping to overcome flooding stress in plants [J]. EMBO J, 2022, 41(19): e112282.
[28]	Liu B, Jiang YZ, Tang H, et al. The ubiquitin E3 ligase SR1 modulates the submergence response by degrading phosphorylated WRKY33 in Arabidopsis [J]. Plant Cell, 2021, 33(5): 1771-1789.
[29]	Wang J, Han MZ, Huang YX, et al. Flooding tolerance of rice: regulatory pathways and adaptive mechanisms [J]. Plants, 2024, 13(9): 1178.
[30]	Hattori Y, Nagai K, Furukawa S, et al. The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water [J]. Nature, 2009, 460(7258): 1026-1030.
[31]	Lin CC, Lee WJ, Zeng CY, et al. SUB1A-1 anchors a regulatory cascade for epigenetic and transcriptional controls of submergence tolerance in rice [J]. PNAS Nexus, 2023, 2(7): pgad229.
[32]	华炫, 田博雯, 周欣彤, 等. 旱柳SmERF B3-45的克隆及耐盐功能研究 [J]. 生物技术通报, 2024, 40(12): 124-135.
	Hua X, Tian BW, Zhou XT, et al. Cloning SmERF B3-45 from Salix matsudana and functional analysis on its tolerance to salt [J]. Biotechnol Bull, 2024, 40(12): 124-135.
[33]	Shang JZ, Song PH, Ma B, et al. Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress [J]. Funct Integr Genomics, 2014, 14(4): 767-777.
[34]	陈发棣, 陈素梅, 宋爱萍, 等. 乙烯调控植物耐涝机制的研究进展 [J]. 南京农业大学学报, 2018, 41(2): 203-208.
	Chen FD, Chen SM, Song AP, et al. Progress in mechanisms of ethylene mediated tolerance of plant to waterlogging [J]. J Nanjing Agric Univ, 2018, 41(2): 203-208.
[35]	Liu NN, Du YH, Yan SJ, et al. The light and hypoxia induced gene ZmPORB1 determines tocopherol content in the maize kernel [J]. Sci China Life Sci, 2024, 67(3): 435-448.
[36]	秦于玲, 刘维侠, 曹振木, 等. 辣椒果实SGR基因的克隆与功能分析 [J]. 热带作物学报, 2025, 46(7): 1562-1570.
	Qin YL, Liu WX, Cao ZM, et al. Cloning and functional analysis of the SGR gene in Capsicum annum fruits [J]. Chin J Trop Crops, 2025, 46(7): 1562-1570.
[37]	马鑫, 雷建峰, 黄诗雨, 等. GhALMT10在干旱胁迫下的功能鉴定 [J]. 棉花学报, 2025, 37(3): 175-184.
	Ma X, Lei JF, Huang SY, et al. Functional identification of GhALMT10 under drought stress [J]. Cotton Sci, 2025, 37(3): 175-184.
[38]	Huang QH, Hua X, Zhang Q, et al. Identification and functional verification of salt tolerance hub genes in Salix matsudana based on QTL and transcriptome analysis [J]. Environ Exp Bot, 2023, 215: 105470.