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

Abstract

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

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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References 41

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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

Overexpressing NPR1 from Arabidopsis thaliana Enhanced Resistance to Fusarium Wilt and Powdery Mildew in Cucumis sativus

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