GmSZFP互作蛋白的筛选及GmERF7在大豆抵抗SMV侵染过程中的功能分析

doi:10.3864/j.issn.0578-1752.2025.14.003

中国农业科学 ›› 2025, Vol. 58 ›› Issue (14): 2739-2750.doi: 10.3864/j.issn.0578-1752.2025.14.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

GmSZFP互作蛋白的筛选及GmERF7在大豆抵抗SMV侵染过程中的功能分析

齐梦楠¹(), 赵玎玲¹, 张雪妍¹, 张玉洁¹, 王荣纳¹, 刘兵强², 闫龙², 张洁¹^,^*(), 王冬梅¹^,^*()

¹ 河北农业大学生命科学学院/华北作物改良与调控国家重点实验室/河北省植物生理与分子病理学重点实验室，河北保定 071001
² 河北省农林科学院粮油作物研究所，石家庄 050035

收稿日期:2025-01-27 接受日期:2025-03-10 出版日期:2025-07-17 发布日期:2025-07-17
通信作者:
张洁，E-mail：zhangjiezhaolh@163.com。
王冬梅，E-mail：dongmeiwang63@126.com。
联系方式: 齐梦楠，E-mail：qmn1486360375@163.com。
基金资助:
国家自然科学基金(32472111); 河北省自然科学基金(C2023204093); 河北省现代农业产业技术体系创新团队建设项目(HBCT2023040101); 河北省现代农业产业技术体系创新团队建设项目(HBCT2023040202); 大豆资源精准鉴定与优异种质创新利用(21326313D-1)

Identification of GmSZFP-Interacting Proteins and Functional Analysis of GmERF7 in Soybean Resistance to SMV Infection

QI MengNan¹(), ZHAO DingLing¹, ZHANG XueYan¹, ZHANG YuJie¹, WANG RongNa¹, LIU BingQiang², YAN Long², ZHANG Jie¹^,^*(), WANG DongMei¹^,^*()

¹ College of Life Sciences, Hebei Agricultural University/State Key Laboratory of North China Crop Improvement and Regulation/ Hebei Provincial Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei
² Institute of Cereals and Oils Crops, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050035

Received:2025-01-27 Accepted:2025-03-10 Published:2025-07-17 Online:2025-07-17

摘要/Abstract

摘要：

【目的】大豆作为一种重要的经济和油料作物，其产量和品质常受大豆花叶病毒病的严重威胁，大豆花叶病毒病由大豆花叶病毒（Soybean mosaic virus，SMV）引发。实验室前期筛选到一个差异表达的C₂H₂型单锌指蛋白基因GmSZFP，在大豆抵抗SMV侵染过程中发挥正调控作用。利用酵母双杂交文库筛选其互作蛋白，并探究互作蛋白在大豆与SMV互作过程中的功能，为进一步解析大豆抵御SMV侵染过程中转录因子的调控网络提供理论依据。【方法】以大豆品种冀豆7号、SMV毒株SC-8和N3组成的亲和、不亲和组合为试验材料，利用酵母双杂交文库筛选GmSZFP的潜在互作蛋白，借助酵母双杂交技术（Y2H）和双分子荧光互补试验（BiFC）验证二者的互作关系。运用实时荧光定量PCR（qPCR）和烟草脆裂病毒（Tobacco rattle virus，TRV）介导的基因沉默技术（TRV-VIGS）分析潜在互作蛋白基因的表达特征及其在大豆与SMV互作过程中的功能。【结果】利用酵母双杂交文库筛选到GmSZFP的潜在互作靶蛋白GmERF7，Y2H和BiFC试验证明二者存在互作关系。GmERF7全长393个AA，包含一个AP2/ERF结构域和2个核定位（NLS）结构域；qPCR结果表明，在亲和组合中，GmERF7在接种病毒后4、12和24 h的表达水平显著高于不亲和组合，且在24 h时表达量达到峰值；利用VIGS技术沉默GmERF7，在亲和组合中，大豆叶片的接种部位有明显的胼胝质积累，而对照中几乎看不到胼胝质荧光；并且在接种后14 d，在基因沉默植株的上位叶中未检测到SMV外壳蛋白基因CP的表达，叶片未出现感染SMV症状，而在对照组中检测到CP的表达，并出现花叶、失绿等感病症状。以上结果表明，沉默GmERF7降低了SMV对大豆植株的侵染能力，增强了大豆对SMV的抗性，说明GmERF7负调控大豆的抗病性。【结论】ERF类转录因子GmERF7能够与锌指蛋白GmSZFP互作，在大豆与SMV互作过程中负调控大豆对SMV的抗性。

关键词: 大豆花叶病毒, GmSZFP, GmERF7, 互作蛋白, 酵母双杂交文库, 双分子荧光互补试验

Abstract:

【Objective】Soybean, an important economic and oil crop, is frequently threatened by soybean mosaic virus (SMV) disease, which is caused by the soybean mosaic virus (SMV). Previous studies identified a differentially expressed C₂H₂ single zinc finger protein gene, GmSZFP, which positively regulated soybean resistance to SMV infection. Using a yeast two-hybrid library, we screened for proteins that interact with GmSZFP and explored their functions in the soybean-SMV interaction. This research provides a theoretical basis for further elucidating the regulatory network of transcription factors involved in soybean resistance to SMV infection. 【Method】In this study, the compatible (Jidou 7 and SMV strain SC-8) and incompatible (Jidou 7 and SMV strain N3) combinations were used to screen the potential interaction proteins of GmSZFP by yeast two-hybrid library. the protein-protein interactions were validated through yeast two-hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC). Real-time quantitative PCR (qPCR) and Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) were employed to analyze the transcriptional expression levels and functions of the identified interaction proteinin the soybean-SMV interaction. 【Result】We identified GmERF7 as apotential target protein of GmSZFP through yeast two-hybrid system. The interaction between GmSZFP and GmERF7 was confirmed by Y2H and BiFC. GmERF7 is a 393 -amino acid protein containing an AP2/ERF domain and two nuclear localization signals (NLS). qPCR results showed that the expression levels of GmERF7 were significantly higher in the compatible combination than those in the incompatible combination at 4, 12, and 24 hours after infection, peaking at 24 hours. VIGS-mediated silencing of GmERF7 in Jidou 7 plants infected with SC-8 (compatible combination) results in increased callose deposition at the inoculation site compared to the control. At the 14 days after inoculation, the expression of SMV coat protein CP gene was undetectable in the upper leaves of the GmERF7-silenced plants, and no SMV infection symptoms were observed. In contrast, the CP gene was detected in the control plants, which exhibited typical susceptibility symptoms such as mosaic and green-loss. These results indicate silencing GmERF7 weakened virus transport and spread between cells, thereby enhancing plant resistance to SMV. This suggest that GmERF7 negatively regulates soybean resistance to SMV. 【Conclusion】The ERF transcription factor GmERF7 interacts with the zinc finger protein GmSZFP, and negatively regulates soybean resistance to SMV.

Key words: Soybean mosaic virus, GmSZFP, GmERF7, interacting proteins, yeast two-hybrid system, BiFC

齐梦楠, 赵玎玲, 张雪妍, 张玉洁, 王荣纳, 刘兵强, 闫龙, 张洁, 王冬梅. GmSZFP互作蛋白的筛选及GmERF7在大豆抵抗SMV侵染过程中的功能分析[J]. 中国农业科学, 2025, 58(14): 2739-2750.

QI MengNan, ZHAO DingLing, ZHANG XueYan, ZHANG YuJie, WANG RongNa, LIU BingQiang, YAN Long, ZHANG Jie, WANG DongMei. Identification of GmSZFP-Interacting Proteins and Functional Analysis of GmERF7 in Soybean Resistance to SMV Infection[J]. Scientia Agricultura Sinica, 2025, 58(14): 2739-2750.

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图/表 8

表1

引物信息"

引物名称Primer name	引物序列Primer sequence (5′-3′)	用途<BOLD>A</BOLD>pplication
GmSZFP编码区-F GmSZFP coding region-F	ATGGTTTCTTTTACAAGTTATT	扩增GmSZFP Amplify GmSZFP
GmSZFP编码区-R GmSZFP coding region-R	TCAATCAATATTATCATCTTCAGA	扩增GmSZFP Amplify GmSZFP
GmERF7编码区-F GmERF7 coding region-F	ATGTGTGGTGGTGCGATTATC	扩增GmERF7 Amplify GmERF7
GmERF7编码区-R GmERF7 coding region-R	TCAGAAGACTCCTGCCATGG	扩增GmERF7 Amplify GmERF7
pGBDT7-GmSZFP-F	gaattcATGGTTTCTTTTACAAGTTATTTC	pGBDT7-GmSZFP载体构建 pGBDT7-GmSZFP vector construction
pGBDT7-GmSZFP-R	ggatccTCAATCAATATTATCATCTTCAGA	pGBDT7-GmSZFP载体构建 pGBDT7-GmSZFP vector construction
pGADT7-GmSZFP-F	catatgTATGGGCCTCTGCACCTCT	pGADT7-GmSZFP载体构建 pGADT7-GmSZFP vector construction
pGADT7-GmSZFP-R	ggatccCCTTGGAAACACTCCGTGGAGTT	pGADT7-GmSZFP载体构建 pGADT7-GmSZFP vector construction
pGADT7-GmERF7-F	catatgATGTGTGGTGGTGCGATTATC	pGADT7-GmERF7载体构建 pGADT7-GmERF7 vector construction
pGADT7-GmERF7-R	gagctcTCAGAAGACTCCTGCCATGG	pGADT7-GmERF7载体构建 pGADT7-GmERF7 vector construction
pGBDT7-GmERF7-F	catatgATGTGTGGTGGTGCGATTATC	pGBDT7-GmERF7载体构建 pGBDT7-GmERF7 vector construction
pGBDT7-GmERF7-R	gagctcTCAGAAGACTCCTGCCATGG	pGBDT7-GmERF7载体构建 pGBDT7-GmERF7 vector construction
pxy104-GmERF7-F	ATTACAGGTACCCGG- ggatccATGTGTGGTGGTGCGATT	pxy104-GmERF7载体构建 pxy104-GmERF7 vector construction
pxy104-GmERF7-R	CACGCTGCCACCGCC- gtcgacGAAGACTCCTGCCATGGA	pxy104-GmERF7载体构建 pxy104-GmERF7 vector construction
pxy106-GmSZFP-F	ATCGAGGACGCCGGC- ggatccATGGTTTCTTTTACAAGTTAT	pxy106-GmSZFP载体构建 pxy106-GmSZFP vector construction
pxy106-GmSZFP-R	GAACGAAAGCTCTGC- gtcgacATCAATATTATCATCTTCAGA	pxy106-GmSZFP载体构建 pxy106-GmSZFP vector construction
qRT-GmERF7-F	AAGCCCTTTGGGTTCTCTCG	检测GmERF7的转录水平表达量 To detect the transcription level of GmERF7
qRT-GmERF7-R	GCATCGTAAGCTCTTGCAGC
EF1b-F	CCACTGCTGAAGAAGATGATGATG	检测EF1b的转录水平表达量 To detect the transcription level of EF1b
EF1b-R	AAGGACAGAAGACTTGCCACTC
pTRV₂-GmERF7-F	ggatccGCTGCTCCTTTGGACGGT	pTRV₂-GmERF7载体构建 pTRV₂ -GmERF7 vector construction
pTRV₂-GmERF7-R	ggtaccTCAGAAGACTCCTGCCATGGA	pTRV₂-GmERF7载体构建 pTRV₂ -GmERF7 vector construction
SMV-CP PCR-F	GAGTGGGACAGGAGCAAAGAGCTTA	检测SMV-CP的表达量 To detect the expression of SMV-CP
SMV-CP PCR-R	TCAAGCAAGTGGTCCAAGCTAAGAA	检测SMV-CP的表达量 To detect the expression of SMV-CP

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编号 Number	序列名 Sequence ID	注释 Annotation	预测功能 Predictive function
1	Glyma.13G145000	苯丙氨酸解氨酶3 Phenylalanine ammonia-lyase 3	在植物生物和非生物胁迫方面发挥作用 Plays a role in plant biotic and abiotic stresses
2	Glyma.06G242900	金属硫蛋白MT3 Metallothionein MT3	在重金属胁迫下诱导表达 Expression was induced under heavy metal stress
3	Glyma.07G044300	乙烯应答转录因子(ERF7) Ethylene Response Transcription Factor (ERF7)	在植物生物和非生物胁迫方面发挥作用 Plays a role in plant biotic and abiotic stresses
4	Glyma.10G251900	多聚泛素 Polyubiquitin	调节蛋白质的降解 Regulates the degradation of proteins
5	Glyma.05G190300	果胶酯酶 Pectin esterase	促进植物细胞壁修饰和随后的分解 Promotes the modification of plant cell walls and subsequent decomposition
6	Glyma.10G090500	腺苷甲硫氨酸 Adenosylmethionine	多种甲基转移酶催化反应的甲基供体；与多种酶的活性相关 Methyl donors for multiple methyltransferase-catalyzed reactions; Associated with the activity of a variety of enzymes
7	Glyma.18G036400	核酮糖二磷酸羧化酶 Ribulose diphosphate carboxylase	激活RuBisCO Activate RuBisCO
8	Glyma.02G147400	跨膜GTPase FZO样 Transmembrane GTPase FZO-like	在类囊体和叶绿体形态的中起作用 Plays a role in thylakoid and chloroplast morphology
9	Glyma.04G003700	异戊二烯化植物蛋白 Isoprenylated plant protein	在重金属胁迫下诱导表达 Expression was induced under heavy metal stress
10	Glyma.13G046200	RBCS1核酮糖二磷酸羧化酶小链1 RBCS1 ribulose diphosphate carboxylase small chain 1	二氧化碳固定中发挥作用 Carbon dioxide fixation

GmSZFP互作蛋白的筛选及GmERF7在大豆抵抗SMV侵染过程中的功能分析

Identification of GmSZFP-Interacting Proteins and Functional Analysis of GmERF7 in Soybean Resistance to SMV Infection

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