利用VIGS分析SmWRKY30在茄子抗青枯病中的作用

doi:10.3864/j.issn.0578-1752.2025.03.011

中国农业科学 ›› 2025, Vol. 58 ›› Issue (3): 548-563.doi: 10.3864/j.issn.0578-1752.2025.03.011

利用VIGS分析SmWRKY30在茄子抗青枯病中的作用

张林琳¹^,²(), 宫瑞¹^,², 崔彦玲², 钟雄辉², 李烨³, 李然红¹(), 潜宗伟²()

¹ 牡丹江师范学院生命科学与技术学院，黑龙江牡丹江 157011
² 北京市农林科学院蔬菜研究所/蔬菜生物育种全国重点实验室/国家蔬菜工程技术研究中心/蔬菜种质改良北京市重点实验室，北京 100097
³ 黑龙江省农业工程职业学院，哈尔滨 150070

收稿日期:2024-06-20 接受日期:2024-09-11 出版日期:2025-02-01 发布日期:2025-02-11
通信作者:
潜宗伟，E-mail：qianzongwei@nercv.org。
李然红，E-mail：swxlrh@126.com
联系方式: 张林琳，E-mail：1614238564@qq.com。
基金资助:
北京市农林科学院科技创新项目(KJCX20230126); 北京市农林科学院蔬菜研究所改革与发展项目(KYCX202301); 北京蔬菜研究中心协同创新(XTCX202301); 山东省重点研发计划（重大科技创新工程）(2021LZGC0017); 国家现代农业产业技术体系资助项目(CARS-23-G-06)

Effect Analysis of SmWRKY30 in Eggplant Resistance to Ralstonia solanacearum by Virus Induced Gene Silencing (VIGS)

ZHANG LinLin¹^,²(), GONG Rui¹^,², CUI YanLing², ZHONG XiongHui², LI Ye³, LI RanHong¹(), QIAN ZongWei²()

¹ College of Life Sciences and Technology, Mudanjiang Normal University, Mudanjiang 157011, Heilongjiang
² Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences/State Key Laboratory of Vegetable Biobreeding/National Engineering Research Center for Vegetables/Beijing Key Laboratory of Vegetable Germplasms Improvement, Beijing 100097
³ Heilongjiang Agricultural Engineering Vocational College, Harbin 150070

Received:2024-06-20 Accepted:2024-09-11 Published:2025-02-01 Online:2025-02-11

摘要/Abstract

摘要：

【目的】转录因子WRKY在植物抗病、抗逆过程中具有重要的调控作用。番茄SlWRKY30是抗青枯病的关键基因，茄子SmWRKY30与番茄SlWRKY30为同源基因。通过青枯菌诱导及基因沉默体系建立，探究茄子SmWRKY30对茄子青枯病的调控作用，为茄子抗青枯病资源的创制和品种选育奠定基础。【方法】以茄子抗病材料YS40和感病材料ZP80为试验材料，克隆SmWRKY30，分析基因、氨基酸和启动子序列差异。青枯菌诱导后，分析2种材料不同组织中SmWRKY30的表达量变化差异。比较茄子与番茄接菌后STH2的表达量变化，分析茄子SmWRKY30与番茄SlWRKY30作用机制的异同。构建基因沉默体系，比较清水组、空载组与试验组植株的病情指数（disease index，DSI）、表型和基因表达量的变化。【结果】SmWRKY30开放阅读框为891 bp，具有WRKY基因家族保守结构域。构建系统进化树发现，SmWRKY30与蒜芥茄和茄子SmWRKY65亲缘关系近。抗病材料YS40和感病材料ZP80的cDNA序列存在6个SNP和1个Indel的差异，氨基酸翻译差异大，启动子区域差异明显，YS40比ZP80少一个GT1-motif启动子元件。接种青枯菌后，分别对抗感病材料根、茎、叶组织的SmWRKY30表达量进行分析，结果显示，接种青枯菌14 d，在根中，YS40中SmWRKY30的表达量显著高于ZP80，而在茎中，SmWRKY30的表达量无明显差异。接种青枯菌5和14 d，在叶片中，YS40中SmWRKY30的表达量显著高于ZP80，表明SmWRKY30的高表达可能与茄子抗青枯病有关。通过对抗病材料YS40进行SmWRKY30沉默，qRT-PCR结果表明，pTRV2::SmWRKY30沉默植株中SmWRKY30的表达量显著低于清水和pTRV2::00空载处理植株，说明抗病材料YS40中SmWRKY30被成功沉默。表型观察发现，接种青枯菌后，pTRV2::SmWRKY30沉默植株叶片明显发黄萎蔫，14 d后DSI为2.07，表现易感青枯病。通过对茄子与番茄同源的4个STH2基因进行定量分析，发现STH2基因表达量均呈先上升后下降的变化趋势。接菌1 d后，抗病材料中仅有STH2-3表达量显著高于感病材料。接菌5 d后，抗病材料中STH2-3和STH2-4的表达量显著低于感病材料。接菌7和14 d后，抗、感病材料中4个STH2基因的表达量均无明显差异。且抗病材料中4个STH2基因的表达情况与SmWRKY30的表达趋势均明显不同。【结论】SmWRKY30可能参与且正向调控茄子抗青枯病相关反应过程，但可能不是通过与STH2基因作用使茄子对青枯病产生抗性。

关键词: 茄子, 青枯病, WRKY转录因子, 病毒诱导的基因沉默, 表达分析

Abstract:

【Objective】The results showed that WRKY transcription factors play an important regulatory role in the process of plant disease resistance and stress resistance. Tomato SlWRKY30 is a key gene for Ralstonia solanacearum resistance, SmWRKY30 was the homologous gene as SlWRKY30 in eggplant. Through the induction of Ralstonia solanacearum and the establishment of gene silencing system, the regulatory effect of SmWRKY30 gene on Ralstonia solanacearum in eggplant was explored, which laid a foundation for the creation of Ralstonia solanacearum resistant resources and variety breeding of eggplant. 【Method】SmWRKY30 was cloned from eggplant disease-resistant material YS40 and disease-susceptible material ZP80 to analyze the differences in genes, amino acids and promoter sequences. After the induction of Ralstonia solanacearum fungus, the differences in the expression of SmWRKY30 in different tissues of the two materials were analyzed. The expression of STH2 in tomato and eggplant was compared, and the similarities and differences between SmWRKY30 and SlWRKY30 were analyzed. Virus-induced gene silencing system was constructed to compare the changes of disease index, phenotype and gene expression in the water group, the empty vector control and the experimental group. 【Result】The SmWRKY30 open reading frame is 891 bp and has a conserved domain of the WRKY gene family, The phylogenetic tree was constructed and it was found that SmWRKY30 was closely related to Solanum sisymbriifolium and Solanum melongena WRKY65. There were 6 SNPs and 1 Indel differences in the cDNA sequences of the disease-resistant material YS40 and the disease-susceptible material ZP80, the amino acid translation difference was large, and the promoter region was significantly different, and YS40 had one lack of GT1-motif promoter element than ZP80. After inoculation with Ralstonia solanacearum and the expression of SmWRKY30 in roots, stems and leaves of disease-susceptible materials were analyzed, the results showed that the expression of SmWRKY30 in YS40 was significantly higher than that of ZP80 in roots after 14 days of inoculation with Ralstonia solanacearum. However, there was no significant difference in the expression of SmWRKY30 in stems. After 5 and 14 days of inoculation with Ralstonia solanacearum, the expression level of SmWRKY30 in YS40 leaves was significantly higher than that of ZP80, indicating that SmWRKY30 high expression may be related to the resistance of Ralstonia solanacearum in eggplant. The qRT-PCR results showed that the expression level of SmWRKY30 in pTRV1/pTRV2::SmWRKY30 silenced plants was significantly lower than that in clean water treatment and pTRV1/pTRV2::00 no-load treatment plants, indicating that SmWRKY30 in the disease-resistant material YS40 was successfully silenced. Phenotypic observation showed that the leaves of pTRV1/pTRV2::SmWRKY30 silenced plants were obviously yellowish wilted, and the disease index was 2.07 at 14 days after inoculation with Ralstonia solanacearum fungus, indicating that they were susceptible to Ralstonia solanacearum. Through the quantitative analysis of four STH2 genes homologous to eggplant and tomato, it was found that the expression levels of STH2 genes increased firstly and then decreased. After 1 day of inoculation, only the expression level of STH2-3 in the disease-resistant materials was significantly higher than that in the disease-susceptible materials. After 5 days of inoculation, the expression levels of STH2-3 and STH2-4 in the disease-resistant materials were significantly lower than those in the disease-susceptible materials. After 7 and 14 days of inoculation, there was no significant difference in the expression levels of the four STH2 genes in the disease-resistant materials and disease-susceptible materials. Moreover, the expression trends of the four STH2 genes in the resistant materials were significantly different from those of SmWRKY30. 【Conclusion】SmWRKY30 may be involved in and positively regulate the response process of Ralstonia solanacearum resistance in eggplant, but may not make eggplant resistant to Ralstonia solanacearum by interacting with STH2 gene.

Key words: eggplant, Ralstonia solanacearum, WRKY transcription factor, VIGS, gene expression analysis

张林琳, 宫瑞, 崔彦玲, 钟雄辉, 李烨, 李然红, 潜宗伟. 利用VIGS分析SmWRKY30在茄子抗青枯病中的作用[J]. 中国农业科学, 2025, 58(3): 548-563.

ZHANG LinLin, GONG Rui, CUI YanLing, ZHONG XiongHui, LI Ye, LI RanHong, QIAN ZongWei. Effect Analysis of SmWRKY30 in Eggplant Resistance to Ralstonia solanacearum by Virus Induced Gene Silencing (VIGS)[J]. Scientia Agricultura Sinica, 2025, 58(3): 548-563.

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引物名称Primer name	序列Sequence (5′-3′)
SmWRKY30-F	ATGGAGAAAGTTAAAGCTTGGGATAAAG
SmWRKY30-R	CTACTCGAAATATTCAGAAATGTCAATCATCAAG
pTRV2-WRKY30-F	GTGAGTAAGGTTACCGAATTCCCTCACAACCATCCATATTCATTT
pTRV2-WRKY30-R	CGTGAGCTCGGTACCGGATCCAATCCGAATGTGCATAGAATGGC
SmActionF	TTCCTTGTATGCTAGTGGTCGTACAA
SmActionR	CTCAGCACCAATGGTAATAACTTGTC
SmWRKY30DLF	GTCTTCTTGTCCATCTATTGTTGACTTG
SmWRKY30DLR	CCCTGTGAGTGCATCTATAATAAGCC
STH2-1F	GAAGATGAATTTTGTTGAAGGTGGTCC
STH2-1R	CACCTTTTGTGTGGTACTCAGTTTTTGTC
STH2-2F	GACCCTAAAAGGCTATTCAAAGCTTTGG
STH2-2R	GTTTCTAGGTTCTTGTCATCAATCACATGAATC
STH2-3F	ATGGGTGTCTTTTCCTATACACATGATGTC
STH2-3R	CATGGATCTTGTGCTTCAAGTACTTGATTG
STH2-4F	ATGGGTGTCACTTCCTACACACATG
STH2-4R	CATCAACAACATGGATCTTGTGCTTCAAG
WRKY30 Q-F	GAAGAATGAAGGATAAACAAATTTGTACCCAC
WRKY30 Q-R	CCAGAGGATCAAACTGGTTTTTTAGCC

材料 <BOLD>M</BOLD>aterial	元件名称 Cis element	核心序列 Sequence	功能 Function	位置 Position (bp)	数目 Number
YS40	GT1-motif	GGTTAA	光响应元件 Light responsive element	209	1
ZP80	GT1-motif	GGTTAA	光响应元件 Light responsive element	209/1006	2

利用VIGS分析SmWRKY30在茄子抗青枯病中的作用

Effect Analysis of SmWRKY30 in Eggplant Resistance to Ralstonia solanacearum by Virus Induced Gene Silencing (VIGS)

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