茄子青枯病抗性相关的E3泛素连接酶基因的筛选及鉴定

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

摘要/Abstract

摘要：

茄子在生产中易受青枯病侵袭。为挖掘茄子青枯病抗性基因，前期以茄子高抗青枯病自交系和感病自交系为试材进行转录组分析，获得4个与调控青枯病抗性相关的E3泛素连接酶基因，分别命名为SmSP1、SmSPL2、SmDDA1a1和SmDDA1a2。序列分析发现，4个E3连接酶的结构域在7个物种中较为保守，SmSP1及SmSPL2属RING型E3连接酶，SmDDA1a1和SmDDA1a2属CRL^DDB型E3的底物受体。亚细胞定位表明，除SmDDA1a2定位于细胞核，SmSP1、SmSPL2和SmDDA1a1定位于细胞核及其他细胞部位。4个基因在茄子抗感材料的根、茎及叶中均有表达，叶中最高。青枯菌及外源激素可诱导4个基因的表达，且在抗性材料中的表达量高于感病材料。水杨酸可诱导SmSP1的表达，茉莉酸甲酯可诱导SmSPL2、SmDDA1a1和SmDDA1a2的表达，乙烯利可诱导4个基因的表达。VIGS基因沉默结果显示，在抗性材料中沉默SmSP1和SmDDA1a2后，导致植株青枯病抗性下降，沉默SmSPL2和SmDDA1a1后，对植株青枯菌抗性没有影响。以上结果表明，SmSP1和SmDDA1a2可能参与茄子青枯病抗性的调控。

关键词: 茄子, 青枯病, E3泛素连接酶, 表达分析, 功能鉴定

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

王亦栖, 颜爽爽, 余炳伟, 甘雨薇, 邱正坤, 朱张生, 陈长明, 曹必好. 茄子青枯病抗性相关的E3泛素连接酶基因的筛选及鉴定[J]. 园艺学报, 2023, 50(10): 2271-2287.

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.

图/表 10

表1 亚细胞定位及VIGS试验引物信息表

Table 1 List of the primers for subcellular localization and VIGS assays

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

表2 qRT-PCR引物信息

Table 2 List of the primers of qRT-PCR

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

图1 茄子高抗和高感自交系（E-31和E-32）接种青枯菌（0 d和7 d）后4个基因的转录组热图（A）及其结构域（B）

Fig. 1 The transcriptome heat map（A）and domain（B）of four genes of eggplant inbred lines with high resistance and high susceptibility（E-31 and E-32）inoculated with Ralstonia solanacearum（0 and 7 days）

图2 茄子 SmSP1、SmSPL2、SmDDA1a1和SmDDA1a2亚细胞定位分析 GFP代表绿色荧光蛋白，NLS代表核定位信号（Sun et al.，2020）。

Fig. 2 Subcellular localization analysis of SmSP1，SmSPL2，SmDDA1a1 and SmDDA1a2 GFP stands for green fluorescent protein，and NLS stands for nuclear localization signal（Sun et al.，2020）.

图3 茄子青枯病高抗和高感自交系（E-31和E-32）幼苗中4个基因表达的组织特异性

Fig. 3 The tissue-specific expression of four genes in eggplant seedlings of bacterial wilt highly resistant and highly susceptible inbred lines（E-31 and E-32） **P < 0.01；* P < 0.05.

图4 茄子高抗和高感自交系（E-31和E-32）接种青枯菌后4个基因的表达量变化显著性差异分析采用LSD多重比较，不同小写字母表示同一材料不同时间差异显著（P < 0.05）。

Fig. 4 The analysis of the expression levels of four genes in eggplant high resistance and high sensitivity inbred lines （E-31 and E-32）inoculated with Ralstonia solanacearum Significant difference analysis was conducted using LSD multiple comparisons，with different lowercase letters indicating significant differences in the same material at different times（P < 0.05）.

图5 茄子高抗青枯病自交系外施水杨酸（SA）、茉莉酸甲酯（MeJA）和乙烯利（Eth）后4个基因的表达变化显著性差异分析采用LSD多重比较，不同小写字母表示同一处理不同时间差异显著（P < 0.05）。

Fig. 5 The expression analysis of four genes in eggplant inbred lines with high resistance to bacterial wilt after applying salicylic acid（SA），methyl jasmonic acid（MeJA）and ethrel（Eth） Significant difference analysis was conducted using LSD multiple comparisons，with different lowercase letters indicating significant differences at different times in the same treatment（P < 0.05）.

图6 茄子高抗自交系VIGS植株的基因表达量及其接种青枯菌后的表型对照表示植株清水处理，pTRV2表示植株注射携带pTRV2空载的农杆菌侵染液。对照及pTRV2处理的误差线表示3个生物学重复差异。显著性差异分析采用LSD多重比较，并用字母标记法（显著性水平为0.01）表示，不同大写字母表示表达量差异显著。

Fig. 6 The expression levels and phenotypes of silenced plants after inoculation with Ralstonia solanacearum The control indicates that the plant is treated with H2O，while pTRV2 indicates that the plant injected with Agrobacterium infection solution carrying pTRV2 empty vector. The error lines of the control and pTRV2 treatment represent three biological repeated differences. Significant difference analysis was conducted using LSD multiple comparisons and represented by letter labeling（significance level 0.01），with different uppercase letters indicating significant differences in expression levels.

表3 茄子高抗自交系VIGS植株接种青枯菌后的发病率和病情指数

Table 3 The statistics list of morbidity and disease index of control and silent plants

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

图7 茄子高抗青枯病自交系VIGS植株中SA信号基因的表达

Fig. 7 The expression of SA signaling genes in silenced plants of eggplant high resistance to bacterial wilt inbred lines ** P < 0.01；* P < 0.05.

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