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

Abstract

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

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

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

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