过表达拟南芥NPR1增强黄瓜对枯萎病和白粉病的抗性

doi:10.3864/j.issn.0578-1752.2023.14.006

摘要/Abstract

摘要：

【背景】黄瓜易感多种病害，特别是枯萎病和白粉病，化学药物防治虽然有效但因残留高和不易降解而被限制使用，培育广谱、持久抗病的黄瓜品种是解决这一难题的根本策略。病程相关基因非表达子1（nonexpressor of pathogenesis- related genes 1，NPR1）是系统获得性抗性（systemic acquired resistance，SAR）中的关键调控因子，参与调控多种防御相关基因的表达，影响植物的抗病能力。【目的】在黄瓜中过表达AtNPR1，探究转基因黄瓜对枯萎病和白粉病的抗性，为培育抗病能力更强、更持久的黄瓜品种提供试验依据。【方法】通过克隆拟南芥AtNPR1，构建AtNPR1过表达载体，利用农杆菌介导法转化黄瓜，获得过表达AtNPR1的转基因黄瓜植株，利用实时荧光定量PCR方法测定转基因植株中相关防御基因的表达量。选择T₀代转基因植株进行枯萎病的抗性鉴定，T₁代转基因植株进行白粉病的抗性鉴定，测定转基因植株接种枯萎病菌（Fusarium oxysporum f. sp. cucumerinum）和白粉病菌（Golovinomyces cichoracearum）后，超氧化物歧化酶（superoxide dismutase，SOD）、过氧化物酶（peroxidase，POD）、过氧化氢酶（catalase，CAT）等抗氧化酶活性变化及丙二醛（malondialdehyde，MDA）、活性氧（reactive oxygen species，ROS）含量的变化。【结果】成功获得8株T₀代转基因植株，OE#4和OE#5为AtNPR1高表达植株，OE#3为AtNPR1低表达植株；通过对转基因植株中相关防御基因的表达量分析发现，过表达AtNPR1的转基因植株中多个相关防御基因表现更强、更快的表达，并且防御基因的表达量与AtNPR1在转基因植株中的表达量呈正相关。其中，PR1、PR4和WRKY70的表达量上调极为显著。转基因植株的抗病性鉴定结果表明，在枯萎病和白粉病的胁迫下，转基因植株表现出更显著的抗性，发病缓慢、症状轻微，病斑面积显著低于野生型（WT）植株。转基因T₀代植株OE#4和OE#5在接种枯萎病菌3 d后未出现明显病斑，接种7 d后出现灰褐色病斑，但未呈现萎蔫等状态；WT植株在接种3 d时出现灰褐色病斑并且轻微萎蔫，7 d时叶片出现严重萎蔫。接种白粉病菌7 d后，T₁代植株OE#2和OE#7和WT植株均出现病斑，但OE#2和OE#7植株病斑面积显著低于WT植株；接种15 d后WT植株出现叶片黄化失绿，而转基因植株OE#2和OE#7病情较轻。与WT植株相比，接种病原菌后，转基因植株中MDA含量较低，SOD、POD、CAT活性保持较高水平，ROS含量累积较少。【结论】黄瓜中过表达AtNPR1可以提高黄瓜对枯萎病和白粉病的抗性。

关键词: 黄瓜, AtNPR1, 枯萎病, 白粉病

Abstract:

【Background】Cucumber (Cucumis sativus) is susceptible to many diseases, especially Fusarium wilt and powdery mildew. Although chemical control is effective, it is limited due to high residue and difficult degradation. Breeding cucumber varieties with broad-spectrum and long-lasting disease resistance is the fundamental strategy to solve this problem. Nonexpressor of pathogenesis-related genes 1 (NPR1) is a key regulator in systemic acquired resistance (SAR), which is involved in regulating the expression of a variety of defense-related genes and affecting plant disease resistance.【Objective】Overexpression of AtNPR1 in cucumber was used to explore the resistance of transgenic cucumber to Fusarium wilt and powdery mildew, and to provide experimental basis for breeding cucumber varieties with stronger and more lasting disease resistance.【Method】The AtNPR1 of Arabidopsis thaliana was cloned, the AtNPR1 overexpression vector was constructed, and the cucumber was transformed by Agrobacterium-mediated method to obtain transgenic cucumber plants with overexpression of AtNPR1. The expression levels of related defense genes in transgenic plants were determined by real-time fluorescence quantitative PCR (qRT-PCR) method. Transgenic plants of T₀ generation were selected for resistance identification of Fusarium wilt, and transgenic plants of T₁ generation were selected for resistance identification of powdery mildew. After inoculation transgenic plants with Fusarium oxysporum f. sp. cucumerinum and Golovinomyces cichoracearum, the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were quantified.【Result】Eight transgenic plants of the T₀ generation were successfully obtained, among which OE#4 and OE#5 exhibited high expression levels of AtNPR1, while OE#3 showed low expression levels. Analysis of the expression level of related defense genes in transgenic plants revealed that those plants overexpressing AtNPR1 exhibited stronger and faster expression of several defense genes. Furthermore, there was a positive correlation between the expression levels of defense genes and AtNPR1 in transgenic plants. Among them, the expression levels of PR1, PR4 and WRKY70 were significantly up-regulated. The results of disease resistance identification of transgenic plants showed that the transgenic plants exhibited more significant resistance, slower onset, mild symptoms, and significantly lower lesion area than wild type (WT) plants when subjected to the stress of Fusarium wilt and powdery mildew. The transgenic T₀ plants OE#4 and OE#5 exhibited no discernible lesions 3 days post-inoculation with F. oxysporum f. sp. cucumerinum, but displayed gray-brown lesions after 7 days without any signs of wilting. Conversely, the WT plants showed gray-brown lesions and slight wilting at 3 d post-inoculation, followed by severe leaf wilting at 7 d. After 7 days of inoculation with G. cichoracearum, both T₁ generation plants OE#2 and OE#7 as well as wild type (WT) plants exhibited lesions. However, the lesion area in OE#2 and OE#7 was significantly smaller than that in WT. After 15 days of inoculation, chlorosis appeared on the leaves of WT plants while the transgenic plants remained mildly affected. Compared with WT plants, transgenic plants exhibited lower MDA content and maintained higher levels of SOD, POD and CAT activities after inoculation. Additionally, the accumulation of ROS was less.【Conclusion】Overexpression of AtNPR1 in cucumber enhances its resistance against Fusarium wilt and powdery mildew.

Key words: cucumber (Cucumis sativus), AtNPR1, Fusarium wilt, powdery mildew

冯向君, 王宏宇, 于静, 池春玉, 丁国华. 过表达拟南芥NPR1增强黄瓜对枯萎病和白粉病的抗性[J]. 中国农业科学, 2023, 56(14): 2701-2712.

FENG XiangJun, WANG HongYu, YU Jing, CHI ChunYu, DING GuoHua. Overexpressing NPR1 from Arabidopsis thaliana Enhanced Resistance to Fusarium Wilt and Powdery Mildew in Cucumis sativus[J]. Scientia Agricultura Sinica, 2023, 56(14): 2701-2712.

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https://www.chinaagrisci.com/CN/Y2023/V56/I14/2701

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引物名称Primer name	序列Sequence (5′-3′)
qPCR-CsPR1-F	TGGAGAAATACGCAAAGGATGG
qPCR-CsPR1-R	CAGGCGGATCATAGTTACAAGTCA
qPCR-CsPR2-F	CCCTCATTCGTTGGGCATTT
qPCR-CsPR2-R	CGGCGTCTTTGTTTGATTGTGA
qPCR-CsPR4-F	ATACGGTTGGACTGCCTTCTGT
qPCR-CsPR4-R	CGAAGCTCCCGTTTCAGTGTTAG
qPCR-CsPR5-F	GGTGCGATGGAGACTTGAGA
qPCR-CsPR5-R	ACTATAAGCAGCAGGGCAAACT
qPCR-CsWRKY70-F	CAAGTCCAGCGACTGCAAGACA
qPCR-CsWRKY70-R	TTGGAGGAGCCGAGCACTATGT
qPCR-CsLOX2-F	AATCAAAGCAGCCTTAGCAACAGC
qPCR-CsLOX2-R	CCTTGACAATGGTGGATGAAGTGA
qPCR-CsAOS-F	AAAAGGATTTGGCTGCTTCTGG
qPCR-CsAOS-R	CACGACCTAATTTGGAATCTACCG
qPCR-CsAPX1-F	TTCGTCTTGCATGGCACTCTG
qPCR-CsAPX1-R	TCCTTGATCGGCTCCAATAGC
qPCR-CsCAT2-F	CTTTCTTGTGCCGATTTCCTTC
qPCR-CsCAT2-R	AAATTGCCCTCCCTGGTGTA