SlSnRK1.2调控番茄抗灰霉病功能分析

doi:10.3864/j.issn.0578-1752.2024.21.006

摘要/Abstract

摘要：

【背景】由灰葡萄孢（Botrytis cinerea）侵染番茄（Solanum lycopersicum）引起的灰霉病严重威胁番茄生产。植物蔗糖非发酵-1-相关蛋白激酶-1（sucrose non-fermenting-1-related protein kinase 1，SnRK1）参与多种植物对生物胁迫和非生物胁迫响应的调控，然而，番茄SnRK1是否参与对灰霉病抗性未见报道。【目的】以灰葡萄孢侵染番茄过程中上调表达的SlSnRK1.2为研究对象，克隆并分析其调控灰霉病抗性功能，为番茄灰霉病防治提供理论依据和基因资源。【方法】采用实时荧光定量PCR（real-time fluorescence quantitative PCR，qRT-PCR）技术，分析SlSnRK1.2在灰葡萄孢侵染番茄过程中以及在番茄不同组织中的表达模式；利用农杆菌介导的瞬时表达技术分析SlSnRK1.2的亚细胞定位情况；借助烟草脆裂病毒（tobacco rattle virus，TRV）介导的基因沉默技术（virus induced gene silencing，VIGS）构建SlSnRK1.2沉默植株，初步分析SlSnRK1.2在番茄与灰葡萄孢互作过程中的作用；利用农杆菌介导的番茄遗传转化体系创制SlSnRK1.2过表达植株，进一步明确SlSnRK1.2在调控番茄对灰霉病抗性中的作用；利用TRV-VIGS技术构建SlSnRK1.2同源基因NbSnRK1.2的沉默植株，分析NbSnRK1.2在烟草与灰葡萄孢互作中的作用。【结果】以Micro-Tom为材料，利用qRT-PCR技术明确SlSnRK1.2的转录表达显著受到灰葡萄孢侵染诱导；亚细胞定位结果显示SlSnRK1.2定位于细胞质和细胞核；qRT-PCR分析表明，SlSnRK1.2在番茄的根、茎、幼嫩叶片、成熟叶片、花蕾和花中均有表达，在茎部的表达量最高；瞬时沉默SlSnRK1.2减弱番茄对灰霉病的抗性，过表达SlSnRK1.2增强番茄对灰霉病的抗性；在此基础上，瞬时沉默SlSnRK1.2的同源基因NbSnRK1.2减弱烟草对灰霉病的抗性。【结论】SlSnRK1.2正调控番茄对灰霉病的抗性，可作为番茄抗灰霉病分子育种的基因资源。

关键词: 番茄灰霉病, 灰葡萄孢, SlSnRK1.2, 抗病性, 病毒介导的基因沉默

Abstract:

【Background】Grey mould, caused by Botrytis cinerea, poses serious threats to tomato production. Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) from plant is involved in the regulation of responses to biotic and abiotic stresses. However, whether tomato SnRK1 is involved in tomato resistance to grey mould remains unclear. 【Objective】In this study, SlSnRK1.2, which was up-regulated in the process of B. cinerea infecting tomato, was used as the research object to clone and analyze its function of regulating grey mould resistance, so as to provide theoretical basis and gene resources for the prevention and control of tomato grey mould. 【Method】The expression patterns of SlSnRK1.2 during the infection stage of B. cinerea and in different tomato tissues were examined through real-time fluorescence quantitative PCR (qRT-PCR); Subcellular localization of SlSnRK1.2 was analyzed using Agrobacterium-mediated transient transformation system; SlSnRK1.2 silencing plants were constructed by tobacco rattle virus (TRV)-mediated gene silencing (VIGS) technology, and the role of SlSnRK1.2 in the interaction between tomato and B. cinerea was preliminarily analyzed. The overexpression plants of SlSnRK1.2 were created by Agrobacterium-mediated tomato genetic transformation system, and the role of SlSnRK1.2 in regulating tomato resistance to grey mould was further clarified. NbSnRK1.2, a homologous gene of SlSnRK1.2, was silenced in N. benthamiana using TRV-mediated gene silencing technology to determine the function of NbSnRK1.2 during the interaction between N. benthamiana and B. cinerea. 【Result】Micro-Tom was used as the wild-type (WT) background, qRT-PCR technology was used to clarify that the transcriptional expression of SlSnRK1.2 was significantly induced by B. cinerea infection. Subcellular localization analysis revealed that SlSnRK1.2 was localized in the cytoplasm and nucleus. qRT-PCR analysis showed that SlSnRK1.2 was expressed in roots, stems, young leaves, mature leaves, flower buds, and flowers of tomato, with the highest relative expression level in the stems. Transient silencing of SlSnRK1.2 attenuated tomato resistance to grey mould, while overexpression of SlSnRK1.2 enhanced tomato resistance to grey mould. On this basis, transient silencing of NbSnRK1.2, a SlSnRK1.2 homologous gene, attenuated tobacco resistance to grey mould. 【Conclusion】SlSnRK1.2 positively regulates tomato resistance to grey mould and can be used as a genetic resource for molecular breeding of tomato resistance to grey mould.

Key words: grey mould of tomato (Solanum lycopersicum), Botrytis cinerea, SlSnRK1.2, disease resistance, virus induced gene silencing (VIGS)

李杰, 梁郅林, 孙燕, 檀根甲, 怀宝玉. SlSnRK1.2调控番茄抗灰霉病功能分析[J]. 中国农业科学, 2024, 57(21): 4238-4247.

LI Jie, LIANG ZhiLin, SUN Yan, TAN GenJia, HUAI BaoYu. Functional Analysis of SlSnRK1.2 in Regulating Tomato Resistance to Grey Mould[J]. Scientia Agricultura Sinica, 2024, 57(21): 4238-4247.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.21.006

https://www.chinaagrisci.com/CN/Y2024/V57/I21/4238

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引物Primer	序列Sequence (5′-3′)	用途Application
SlSnRK1.2-RT-F	GGAAACATTATGCGAGAT	实时荧光定量PCR qRT-PCR
SlSnRK1.2-RT-R	AGGTGACTTGGAAGGGTGTAG
SlActin-RT-F	TCTTTCCAATCATTGTGGTGCCTCC
SlActin-RT-R	GAGCCTCCAATCCAGACAC
BcActin-RT-F	TCCAAGCGTGGTATTCTTACCC
BcActin-RT-R	TGGTGCTACACGAAGTTCGTTG
SlSnRK1.2-VIGS-F	TAAGGTTACCGAATTCATGAGTTCCAGAGGTGGTGG	基因沉默 VIGS
SlSnRK1.2-VIGS-R	GCTCGGTACCGGATCCCCCAGACTTGACATACTCCA	基因沉默 VIGS
SlSnRK1.2-GFP-F	GGGACTCTAGAGGATCCATGAGTTCCAGAGGTGGTGG	亚细胞定位 Subcellular localization
SlSnRK1.2-GFP-R	GCTCGAATTCGGATCCTCTTGTGGCCCCTCTAGCTGCC	亚细胞定位 Subcellular localization
OE-SlSnRK1.2-F	ACACGGGGGACTTTGCAACATGAGTTCCAGAGGTGGTG	番茄过表达载体构建 Overexpression vector construction of tomato
OE-SlSnRK1.2-R	CCTCGCCCTTCACGATACATTGTGGCCCCTCTAGCTGC	番茄过表达载体构建 Overexpression vector construction of tomato
NbSnRK1.2-VIGS-F	TAAGGTTACCGAATTCCCATGGATCCTTTGGTAAAG	基因沉默 VIGS
NbSnRK1.2-VIGS-R	GCTCGGTACCGGATCCATCTGCTGGAAAAAATGGCG	基因沉默 VIGS
NbActin-F	ACCAGATTAATGAGCCCAAGAG	实时荧光定量PCR qRT-PCR
NbActin-R	CCAACAGGGACAGTACCAATAC
NbSnRK1.2-RT-F	TTACAGCCACAAACAGCTGTG
NbSnRK1.2-RT-R	TCTTTCCAATCATTGTGGTGCCTCC