Screening and Identification of E3 Ubiquitin Ligase Genes Relate to Bacterial Wilt Resistance in Eggplant

doi:10.16420/j.issn.0513-353x.2022-0835

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (10): 2271-2287.doi: 10.16420/j.issn.0513-353x.2022-0835

• Plant Protection • Previous Articles Next Articles

Screening and Identification of E3 Ubiquitin Ligase Genes Relate to Bacterial Wilt Resistance in Eggplant

WANG Yixi, YAN Shuangshuang, YU Bingwei, GAN Yuwei, QIU Zhengkun, ZHU Zhangsheng, CHEN Changming, CAO Bihao^*()

Key Laboratory of Biology & Genetic Improvement of Horticultural Crops，Ministry of Agriculture and Rural Affairs，Guangdong Vegetable Engineering & Technology Research Center，College of Horticulture，South China Agricultural University，Guangzhou 510642，China

Received:2023-04-28 Revised:2023-05-09 Online:2023-10-25 Published:2023-10-31

Abstract

Abstract:

Eggplant is vulnerable to bacterial wilt in production. In order to investigate eggplant bacterial wilt resistance genes，previously，the transcriptome of highly resistant inbred line and susceptible inbred line were performed and analyzed. Four E3 ubiquitin ligase genes related to bacterial wilt resistance，including SmSP1，SmSPL2，SmDDA1a1，and SmDDA1a2 were obtained. Sequence analysis showed that the domains of four E3 ligase proteins were conservative among seven species. SmSP1 and SmSPL2 belonged to RING type E3 ligase，SmDDA1a1 and SmDDA1a2 belonged to CRL^DDBtype E3 substrate receptor. The subcellular localization results indicated that SmDDA1a2 was localized in the nucleus，and SmSP1，SmSPL2 and SmDDA1a1 were localized in the nucleus and other cell sites. The four genes were expressed in the roots，stems and leaves of eggplant，and the highest expression was found in the leaves. The expression of four genes were induced by Ralstonia solanacearum and exogenous hormones，and higher in resistant materials than in susceptible materials. SmSP1 was induced by salicylic acid，SmSPL2，SmDDA1a1 and SmDDA1a2 were induced by methyl jasmonate，and all four genes were induced by ethephon. Function identification by VIGS demonstrated that silencing of SmSP1 and SmDDA1a2 in resistant materials led to the decline of plant resistance to bacterial wilt，and silencing of SmSPL2 and SmDDA1a1 in resistant materials did not affect the plant resistance to bacterial wilt. These results suggested that SmSP1 and SmDDA1a2 may be involved in the regulation of eggplant bacterial wilt resistance.

Key words: eggplant, bacterial wilt, E3 ubiquitin ligase, expression analysis, functional identification

WANG Yixi, YAN Shuangshuang, YU Bingwei, GAN Yuwei, QIU Zhengkun, ZHU Zhangsheng, CHEN Changming, CAO Bihao. Screening and Identification of E3 Ubiquitin Ligase Genes Relate to Bacterial Wilt Resistance in Eggplant[J]. Acta Horticulturae Sinica, 2023, 50(10): 2271-2287.

Figures/Tables 10

References 63

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名称 Name	序列（5′-3′） Sequence	质粒 Plasmid	酶切位点 Restriction site
SmSP1-GFP	F：ctgcccaaattcgcgaccggt ATGGCTCCATGGGGCGGA	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt ATGGCGAAAAGTTTTCACA	pEAQ-EGFP	Age I
SmSPL2-GFP	F：ctgcccaaattcgcgaccggt ATGTCAATACACGACCAAGCGG	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt AGAATCATATATCCTTACGGAACT	pEAQ-EGFP	Age I
SmDDA1a1-GFP	F：ctgcccaaattcgcgaccggtATGGGGTCAATTTTCGGTGAA	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt GGTGGACACCTTAAGATGCT	pEAQ-EGFP	Age I
SmDDA1a2-GFP	F：ctgcccaaattcgcgaccggt ATGGGGTCAATTTTCGGTGAA	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt GGTGGACACCTTAAGATGCT	pEAQ-EGFP	Age I
pEAQ-EGFP-Universal	F：AGAGTTTTCCCGTGGTTTTCGAACT
	R：GGACACGCTGAACTTGTGGCCGTTT
pTRV2-SmSP1	F：gtgagtaaggttaccgaattc ATGGCTCCATGGGGCGGA	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAATGGCGAAAAGTTTTCACA	pTRV2	BamH I
pTRV2-SmSPL2	F：gtgagtaaggttaccgaattc ATGTCAATACACGACCAAGCGG	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAAGAATCATATATCCTTACGGA	pTRV2	BamH I
pTRV2-SmDDA1a1	F：gtgagtaaggttaccgaattc ATGGGGTCAATTTTCGGTGAA	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAGGTGGACACCTTAAGATGCT	pTRV2	BamH I
pTRV2-SmDDA1a2	F：gtgagtaaggttaccgaattc ATGGGGTCAATTTTCGGTGAA	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAGGTGGACACCTTAAGATGCT	pTRV2	BamH I
pTRV2-Universal	F：TGAGGGAAAAGTAGAGAACG
	R：CCTATGGTAAGACAATGAGT

名称 Name	序列（5′-3′） Sequence	质粒 Plasmid	酶切位点 Restriction site
SmSP1-GFP	F：ctgcccaaattcgcgaccggt ATGGCTCCATGGGGCGGA	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt ATGGCGAAAAGTTTTCACA	pEAQ-EGFP	Age I
SmSPL2-GFP	F：ctgcccaaattcgcgaccggt ATGTCAATACACGACCAAGCGG	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt AGAATCATATATCCTTACGGAACT	pEAQ-EGFP	Age I
SmDDA1a1-GFP	F：ctgcccaaattcgcgaccggtATGGGGTCAATTTTCGGTGAA	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt GGTGGACACCTTAAGATGCT	pEAQ-EGFP	Age I
SmDDA1a2-GFP	F：ctgcccaaattcgcgaccggt ATGGGGTCAATTTTCGGTGAA	pEAQ-EGFP	Age I
	R：gcccttgctcaccataccggt GGTGGACACCTTAAGATGCT	pEAQ-EGFP	Age I
pEAQ-EGFP-Universal	F：AGAGTTTTCCCGTGGTTTTCGAACT
	R：GGACACGCTGAACTTGTGGCCGTTT
pTRV2-SmSP1	F：gtgagtaaggttaccgaattc ATGGCTCCATGGGGCGGA	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAATGGCGAAAAGTTTTCACA	pTRV2	BamH I
pTRV2-SmSPL2	F：gtgagtaaggttaccgaattc ATGTCAATACACGACCAAGCGG	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAAGAATCATATATCCTTACGGA	pTRV2	BamH I
pTRV2-SmDDA1a1	F：gtgagtaaggttaccgaattc ATGGGGTCAATTTTCGGTGAA	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAGGTGGACACCTTAAGATGCT	pTRV2	BamH I
pTRV2-SmDDA1a2	F：gtgagtaaggttaccgaattc ATGGGGTCAATTTTCGGTGAA	pTRV2	EcoR I
	R：cgtgagctcggtaccggatcc TCAGGTGGACACCTTAAGATGCT	pTRV2	BamH I
pTRV2-Universal	F：TGAGGGAAAAGTAGAGAACG
	R：CCTATGGTAAGACAATGAGT

用途 Application	名称 Name	序列（5′-3′） Sequence
表达组织特异性分析 Expression tissue specificity analysis 青枯菌/激素处理 R. solanacearum/hormone treatment VIGS实验 VIGS assay	qPCR-SmSP1	F：CAAGTATGCTTCAATGGGTT
		R：GAGGTCCTTTCTATTATCTGCT
	qPCR-SmSPL2	F：CATCCCCTACCTCTTGTG
		R：AAATCCCATCCTTTGAGC
	qPCR-SmDDA1a1	F：ATCAAGGAAATGCAGGCA
		R：TGGACACCTTAAGATGCT
	qPCR-SmDDA1a2	F：CGCCTGTGACTTATCGCCCTAC
		R：CTCGCTTTGGTCTCAACTTCTC
SA途径信号基因表达 SA pathway signaling gene expression	qPCR-TAG	F：GCAAGTGACCCTGAACTACGAAG
		R：GGGTTTTCCACATCCCTGACAAG
	qPCR-NPR1	F：CTTGGACTGGGTGTTGCTAATG
		R：TGCCCATCCAATGTAATGTCTG
	qPCR-EDS1	F：GTTTCGCAGACAAGTTGAGCC
		R：CTCTGTGTGAACCGATAACGC
	qPCR-PAD4	F：ACATCGGCTGAAACCTCCTTATT
		R：TTTGATAAGTGGTGGGGAAATGA
	qPCR-SGT1	F：TTCTCGGTTTTGAGGAAGGG
		R：GCAGATACCAAGTGATGTCTACCA
	qPCR-GluA	F：GCCGACTGGGTGAGATGGTAA
		R：ACATTGTTGTGCCCGTGGAC
内参基因 Internal reference gene	qPCR-SmActin	F：GTCGGAATGGGACAGAAGGATG
内参基因 Internal reference gene		R：GTGCCTCAGTCAGGAGAACAGGGT

用途 Application	名称 Name	序列（5′-3′） Sequence
表达组织特异性分析 Expression tissue specificity analysis 青枯菌/激素处理 R. solanacearum/hormone treatment VIGS实验 VIGS assay	qPCR-SmSP1	F：CAAGTATGCTTCAATGGGTT
		R：GAGGTCCTTTCTATTATCTGCT
	qPCR-SmSPL2	F：CATCCCCTACCTCTTGTG
		R：AAATCCCATCCTTTGAGC
	qPCR-SmDDA1a1	F：ATCAAGGAAATGCAGGCA
		R：TGGACACCTTAAGATGCT
	qPCR-SmDDA1a2	F：CGCCTGTGACTTATCGCCCTAC
		R：CTCGCTTTGGTCTCAACTTCTC
SA途径信号基因表达 SA pathway signaling gene expression	qPCR-TAG	F：GCAAGTGACCCTGAACTACGAAG
		R：GGGTTTTCCACATCCCTGACAAG
	qPCR-NPR1	F：CTTGGACTGGGTGTTGCTAATG
		R：TGCCCATCCAATGTAATGTCTG
	qPCR-EDS1	F：GTTTCGCAGACAAGTTGAGCC
		R：CTCTGTGTGAACCGATAACGC
	qPCR-PAD4	F：ACATCGGCTGAAACCTCCTTATT
		R：TTTGATAAGTGGTGGGGAAATGA
	qPCR-SGT1	F：TTCTCGGTTTTGAGGAAGGG
		R：GCAGATACCAAGTGATGTCTACCA
	qPCR-GluA	F：GCCGACTGGGTGAGATGGTAA
		R：ACATTGTTGTGCCCGTGGAC
内参基因 Internal reference gene	qPCR-SmActin	F：GTCGGAATGGGACAGAAGGATG
内参基因 Internal reference gene		R：GTGCCTCAGTCAGGAGAACAGGGT

Screening and Identification of E3 Ubiquitin Ligase Genes Relate to Bacterial Wilt Resistance in Eggplant

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沉默基因 Silence gene	总数/株 Total	发病株数/株 Disease plants	发病率/% Morbidity	发病等级Evaluation of scale					病情指数 Disease index
沉默基因 Silence gene	总数/株 Total	发病株数/株 Disease plants	发病率/% Morbidity	0	1	2	3	4	病情指数 Disease index
SmSP1	10	8	80	2	0	1	3	4	67.5
SmSPL2	10	5	50	5	1	2	1	1	30.0
SmDDA1a1	10	4	40	6	1	2	1	0	20.0
SmDDA1a2	20	15	75	5	2	3	8	2	50.0
对照Control（H₂O）	10	0	0	10	0	0	0	0	0
对照Control（pTRV2）	10	0	0	10	0	0	0	0	0

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