α-苦瓜素基因提高番茄对烟草花叶病毒抗性的机理研究

doi:10.16420/j.issn.0513-353x.2023-0062

摘要/Abstract

摘要：

为了深入探究α-苦瓜素基因（αMMC）抗烟草花叶病毒（TMV）的机理，利用“番茄—TMV”互作系统，分析了αMMC转基因植株和野生型植株的生理生化参数及防御相关基因的表达。结果表明，αMMC为Ⅰ型核糖体失活蛋白，定位于细胞质；αMMC可以抑制病毒在寄主体内的繁殖，减轻番茄的病害症状，正调控番茄的防御系统。TMV侵染引起寄主体内活性氧的爆发，αMMC可以增强抗氧化酶的活性以清除活性氧，降低植物的氧化损伤，维持细胞内的氧化平衡。RT-qPCR检测结果表明，αMMC能特异性诱导防御相关基因PR1、PR4、PR6和PRB1-3的表达以提高寄主抵御TMV侵染的能力。

关键词: 番茄, α-苦瓜素, TMV, 抗性机理, 氧化损伤

Abstract:

To explore the mechanism of α-momordicin（αMMC）resistance to tobacco mosaic virus（TMV），the physiological parameters，virus accumulation，and the transcript levels of pathogenesis- related protein（PR）gene in αMMC transgenic plants and wild-type plants under TMV inoculation were analyzed using the‘tomato-TMV’interaction system. Subcellular localization and phenotypic analysis showed that αMMC was a type I ribosome-inactivating protein and localized in the cytoplasm. Furthermore，αMMC could inhibit virus accumulation in the host and alleviate the disease symptoms in tomato，positively the plant’s defense system. Oxidative damage and enzyme activity assays showed that TMV infection caused an outbreak of reactive oxygen species，and αMMC could enhance the activities of antioxidant enzyme to remove excessive reactive oxygen species，resulting in less oxidative damage. In addition，real-time quantitative PCR results indicated that αMMC could specifically induce the expression level of defense-related genes PR1，PR4，PR6 and PRB1-3 to improve host resistance to TMV infection.

Key words: tomato, α-momorcharin, tobacco mosaic virus, resistance mechanism, oxidative damage

杨婷, 席德慧, 夏明, 李佳楠. α-苦瓜素基因提高番茄对烟草花叶病毒抗性的机理研究[J]. 园艺学报, 2024, 51(5): 1126-1136.

YANG Ting, XI Dehui, XIA Ming, LI Jianan. Studies on the Mechanism of α-Momordicin Gene Enhancing Tomato Resistance to Tobacco Mosaic Virus[J]. Acta Horticulturae Sinica, 2024, 51(5): 1126-1136.

图/表 8

表1 本试验中荧光定量 PCR 引物序列

Table 1 Primers used for real-time PCR analysis in this study

引物名称 Primer	引物序列（5′-3′） Primer sequence	基因序列号 Gene ID
Actin-F	CACCATTGGGTCTGAGCAT	NM_001330119
Actin-R	GGGCGACAACCTTGATCTTC	NM_001330119
TMV-F	TAGACCCGCTAGTCACAG	HE818460.1
TMV-R	CAGAGGTCCAAACCAAAC	HE818460.1
PR1-F	TCCGTGCTGGAGGCTTAT	LT856234
PR1-R	AGGCAACTGGACCGA	LT856234
PR4-F	CAGTTAATTTGTGGGTAGG	LOC101257309
PR4-R	CAGGTGGGTAATAGTTGC	LOC101257309
PR5-F	TGAATGTAGTGGGCTTGG	LOC101259255
PR5-R	TGACGCCTTGAGGTAGAG	LOC101259255
PR6-F	CTCACAACTCAGCACGAT	LOC101257608
PR6-R	CCACTTCCCTTAGCAATA	LOC101257608
PRB1-3-F	CCACTCAAATGGAGCAAT	LOC101264239
PRB1-3-R	TCTTTCGCCCACATCGTC	LOC101264239
STH-2-F	GAATGTGAAAATGGAGGGT	LOC101246666
STH-2-R	AGGCAAAAGGATTAGCAAG	LOC101246666

图1 αMMC在本氏烟的亚细胞定位

Fig. 1 Subcellular localization of αMMC in Nicotiana benthamiana

图2 番茄αMMC转基因株系#1和#8接种TMV后幼苗（A）和新叶（B）的表型

Fig. 2 Phenotypes of seedlings（A）and new leaves（B）of αMMC transgenic lines #1 and #8 inoculated with TMV

图3 番茄αMMC转基因株系#1和#8接种TMV后新叶叶面积、叶绿素含量和病毒外壳蛋白基因表达量不同小写字母代表差异显著（双因素方差分析，P < 0.05）。下同。

Fig. 3 Leaf area，chlorophyll content and expression level of viral coat protein in αMMC transgenic lines #1 and #8 inoculated with TMV Different lowercase letters indicate significant differences（two-way ANOVA，P < 0.05）. The same below.

图4 番茄αMMC转基因株系#1和#8接种TMV后叶片中的ROS的积累水平

Fig. 4 ROS accumulation in leaves of αMMC transgenic lines #1 and #8 inoculated with TMV

图5 番茄αMMC转基因植株#1和#8接种TMV后叶片中的丙二醛含量、相对电导率和相对含水量

Fig. 5 The malondialdehyde content，electrolyte leakage and relative water content in leaves of αMMC transgenic plants #1 and #8 inoculated with TMV

图6 番茄αMMC转基因植株#1和#8接种TMV后叶片中的抗氧化酶活性

Fig. 6 The antioxidant enzyme activity in leaves of αMMC transgenic plants #1 and #8 inoculated with TMV

图7 叶片中防御相关基因PR的表达模式

Fig. 7 Functional analysis of PR genes in response to TMV infection in tomato leaves

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