葡萄抗灰霉病相关转录因子VviERF045下游靶基因的筛选

doi:10.3864/j.issn.0578-1752.2025.13.007

摘要/Abstract

摘要：

【背景】灰霉病是严重危害葡萄（Vitis vinifera）产业的重要真菌性病害。乙烯响应因子（AP2/ERF）作为植物中重要的转录因子家族，在调控植物生长发育及逆境响应过程中发挥关键作用。【目的】探究葡萄乙烯响应因子VviERF045响应灰葡萄孢（Botrytis cinerea）侵染的分子机制，通过对其靶基因进行预测并分析其调控作用，为后续进一步解释葡萄抗灰霉病的分子机制及培育抗病新品种提供参考。【方法】采用琼脂盘接种法和孢子悬浮液接种法对葡萄叶片进行灰葡萄孢接种，并利用实时荧光定量PCR（qRT-PCR）技术分析VviERF045及其预测靶基因的表达水平变化；通过PlantCARE在线网站预测该基因启动子区的顺式作用元件；采用MEGA 7和DNAMAN软件对VviERF045蛋白进行系统发育树构建及序列比对分析。利用农杆菌介导的葡萄‘赤霞珠’叶片瞬时转化技术，验证VviERF045在灰葡萄孢侵染过程中的功能；以欧洲葡萄‘黑比诺’叶片为试验材料，于接种灰葡萄孢12 h后，利用DAP亲和纯化测序（DAP-seq）技术在全基因组水平进行分析识别并结合的DNA元件和下游基因。【结果】农杆菌介导的瞬时转化试验发现，在欧洲葡萄‘赤霞珠’叶片中过表达VviERF045显著增强了对灰霉病抗性；对欧洲葡萄‘黑比诺’叶片接菌12 h时进行DAP-seq分析，经两次试验组的信号峰比对发现，共51 806个重复信号峰，对这些信号峰进行promoter-TSS上下2 kb区域的基因筛选，并选取显著性前2 000的基因进行GO和KEGG功能注释。对潜在靶基因预测及信号峰分析，最终预测出3个潜在靶基因（TCP8、SAP5、bHLH48），其启动子区域均呈现显著结合峰；利用qRT-PCR进行验证，其表达水平均出现上调，表明这3个潜在靶基因可能与VviERF045共同参与对灰葡萄孢的响应过程。【结论】 VviERF045在葡萄应对灰葡萄孢侵染过程中起正调控作用，预测TCP8、SAP5、bHLH48可能共同参与灰葡萄孢调控网络。研究结果可为挖掘欧洲葡萄抗病基因提供参考。

关键词: 欧洲葡萄, VviERF045, 灰葡萄孢, 灰霉病, 靶基因, 瞬时转化, DAP亲和纯化测序

Abstract:

【Background】 Gray mold is an important fungal disease that seriously endangers the grapevine (Vitis vinifera) industry worldwide. As an important transcription factor family in plants, ethylene-responsive factor (AP2/ERF) family plays a key role in regulating plant growth and development and stress response. 【Objective】 The study aimed to elucidate the molecular mechanisms by which the grapevine ethylene-responsive factor VviERF045 mediates defense against Botrytis cinerea. Through prediction and functional analysis of its downstream target genes, this research provides new insights into the transcriptional regulatory network associated with grapevine resistance to B. cinerea, establishing a foundation for breeding disease-resistant cultivars. 【Method】 Grapevine leaves were inoculated with B. cinerea using both agar disc and spore suspension methods. The expression profiles of VviERF045 and its predicted targets were evaluated via quantitative real-time PCR (qRT-PCR). Promoter cis-acting elements were analyzed using PlantCARE, while phylogenetic relationships and sequence alignments of VviERF045 were assessed using MEGA 7 and DNAMAN. Functional validation was conducted through Agrobacterium-mediated transient overexpression of VviERF045 in ‘Cabernet Sauvignon’ leaves. To further explore its regulatory landscape, DNA affinity purification sequencing (DAP-seq) was performed on ‘Pinot Noir’ leaves at 12 hours post-inoculation (hpi) with B. cinerea. 【Result】 Transient overexpression of VviERF045 significantly enhanced resistance to gray mold in ‘Cabernet Sauvignon’ leaves. DAP-seq analysis was performed on V. vinifera ‘Pinot Noir’ leaves at 12 hpi. Comparative analysis of peaks between two experimental replicates identified 51 806 consensus peaks. Subsequent genomic annotation revealed these peaks were predominantly located within ± 2 kb regions flanking promoter-transcription start sites (TSS). The top 2 000 most statistically significant peaks were selected for functional characterization through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Through integrated analysis of potential target genes and peak calling, three high-confidence candidate target genes (TCP8, SAP5, and bHLH48) were identified, all showing significant binding peaks in their promoter regions. Subsequent qRT-PCR validation confirmed the transcriptional upregulation of these genes, suggesting their cooperative involvement with VviERF045 in the grapevine’s response to B. cinerea infection.【Conclusion】VviERF045 functions as a positive regulator of grapevine defense against B. cinerea, likely by activating key stress-responsive genes such as TCP8, SAP5, and bHLH48. These results provide mechanistic insights into the pathogen-responsive transcriptional network in grapevine and identify potential molecular targets for breeding resistant V. vinifera cultivars.

Key words: Vitis vinifera, VviERF045, Botrytis cinerea, gray mold, target gene, transient transformation, DAP-seq

赵钰磊, 辛佳璐, 李承男, 李珊, 谢旭飞, 尹晓. 葡萄抗灰霉病相关转录因子VviERF045下游靶基因的筛选[J]. 中国农业科学, 2025, 58(13): 2578-2590.

ZHAO YuLei, XIN JiaLu, LI ChengNan, LI Shan, XIE XuFei, YIN Xiao. Screening of Target Genes Downstream of VviERF045, a Transcription Factor Associated with Gray Mold Resistance in Vitis vinifera[J]. Scientia Agricultura Sinica, 2025, 58(13): 2578-2590.

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NCBI登录号 NCBI accession number	注释 Description	引物序列 Primer sequence
XP_002272228.1	转录因子TCP8 Transcription factor TCP8	F: GCTTCACTCAACCTCGTCACTCC
XP_002272228.1	转录因子TCP8 Transcription factor TCP8	R: AGGACGCAGGCTTAGAGGATGTAG
XP_003633555.1	A20/AN1型锌指胁迫相关蛋白5 Zinc finger A20 and AN1 domain-containing stress-associated protein 5	F: GGTGAATCGGTGCTCTGGATGC
XP_003633555.1		R: CGGCGGTCTTGTAATCGTAGCTG
XP_002282897.1	转录因子bHLH48 Transcription factor bHLH48	F: GCGAAAGGAGCGAGAGAAGAAGG
XP_002282897.1	转录因子bHLH48 Transcription factor bHLH48	R: GCTATGGCTATCAGTGGCTTGGC
XP_002276899.1	含LOB结构域蛋白41 LOB domain-containing protein 41	F: TGCGGATGAGTTGTAATGGCTGTC
XP_002276899.1	含LOB结构域蛋白41 LOB domain-containing protein 41	R: GCGAGGAAGACGGTGGCATTG
KX179904.1	乙烯转录因子ERF045，ENTAV115 Ethylene transcription factor (ERF045)	F: TCTGAAATTCGCCACCCACTTCTG
KX179904.1		R: TTCTCGCCCTCGGACCACAC
VIT_12s0178g00200	内参 Actin	F: AACCCCAAGGCCAACAGAGAAAA
VIT_12s0178g00200	内参 Actin	R: CACCATCACCAGAATCCAGCACA

元件名称 Element name	序列 Sequence	元件数量 Number of elements	功能 Function
ATCT-motif	AATCTAATCC	1	光响应元件Light-responsive element
Gap-box	CAAATGAA（A/G）A	1	光响应元件Light-responsive element
GARE-motif	TCTGTTG	1	赤霉素响应元件Gibberellin-responsive element
GATT-motif	CTCCTGATTGGA	1	光响应元件Light-responsive element
GT1-motif	GGTTAA	1	光响应元件Light-responsive element
I-box	TGATAATGT	1	光响应元件Light-responsive element
TCA-element	TCAGAAGAGG	2	水杨酸响应元件Salicylic acid-responsive element
TC-rich repeats	ATTCTCTAAC	1	防御和胁迫响应元件Defence and stress-responsive element
TCT-motif	TCTTAC	1	光响应元件Light-responsive element

基因名称 Gene name	基因编号 Gene ID	P值 P value	FDR	蛋白名称 Uniprot_ID	注释 Description
LOC100266605	XP_002272228.1	4.42E-133	2.33E-128	F6GYS5	转录因子TCP8 Transcription factor TCP8
LOC100852428	XP_003633555.1	2.01E-125	7.16E-121	F6HBB0	A20/AN1型锌指胁迫相关蛋白5 Zinc finger A20 and AN1 domain-containing stress-associated protein 5
LOC100241856	XP_002282897.1	7.67E-121	2.14E-116	D7TI19	转录因子bHLH48 Transcription factor bHLH48