番茄SlN-like的克隆、表达与抗病毒功能

doi:10.3864/j.issn.0578-1752.2021.20.009

中国农业科学 ›› 2021, Vol. 54 ›› Issue (20): 4348-4357.doi: 10.3864/j.issn.0578-1752.2021.20.009

番茄SlN-like的克隆、表达与抗病毒功能

刘昌云¹(),李欣羽¹,田绍锐¹,王靖¹,裴悦宏¹,马小舟^1,²,樊光进¹,汪代斌³(),孙现超¹()

¹西南大学植物保护学院,重庆 400715
²西南大学园艺园林学院南方山地园艺学教育部重点实验室,重庆 400715
³重庆烟草科学研究所,重庆 400715

收稿日期:2021-04-02 接受日期:2021-04-24 出版日期:2021-10-16 发布日期:2021-10-25
通讯作者: 汪代斌,孙现超
作者简介:刘昌云,E-mail： 15228920380@163.com。
基金资助:
国家自然科学基金(31870147);国家自然科学基金(31670148);西南大学大学生创新创业训练计划(X202010635495);中国烟草总公司重庆公司科技项目(A20201NY02-1306);中国烟草总公司重庆公司科技项目(B20211-NY1315);中国烟草总公司重庆公司科技项目(B20202NY1338)

Cloning, Expression and Anti-Virus Function Analysis of Solanum lycopersicum SlN-like

LIU ChangYun¹(),LI XinYu¹,TIAN ShaoRui¹,WANG Jing¹,PEI YueHong¹,MA XiaoZhou^1,²,FAN GuangJin¹,WANG DaiBin³(),SUN XianChao¹()

¹College of Plant Protection, Southwest University, Chongqing 400715
²Key Laboratory of Horticulture Science for Southern Mountainous Regions of Ministry of Education, College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715
³Chongqing Tobacco Science Research Institute, Chongqing 400715

Received:2021-04-02 Accepted:2021-04-24 Online:2021-10-16 Published:2021-10-25
Contact: DaiBin WANG,XianChao SUN

摘要/Abstract

摘要：

【目的】番茄（Solanum lycopersicum）作为重要的蔬菜作物,其生长受到包括害虫、真菌、细菌和病毒等各种生物因素的危害。明确番茄抗性基因SlN-like的抗病毒功能与机制,为番茄的抗病毒育种与抗病毒药剂的靶向开发提供理论依据。【方法】从茄科植物基因组数据库Solanaceae Genomics Network中获得SlN-like的全长,并将其分为4段,利用融合聚合酶链式反应（fusion PCR）扩增获得SlN-like的序列全长;通过生物信息学分析SlN-like的进化关系、蛋白特征、保守结构域、亚细胞定位以及互作关系;通过实时荧光定量PCR分析SlN-like在番茄根、茎、叶、花和果实中的表达情况及其在烟草花叶病毒（tobacco mosaic virus,TMV）侵染后的叶片表达量;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）沉默番茄内源SlN-like,摩擦接种TMV-GFP于沉默植株,明确SlN-like对病毒侵染的影响。实时荧光定量PCR分析沉默植株中脱落酸（abscisic acid）、茉莉酸（jasmonic acid）和乙烯（ethylene）激素相关基因的表达量及SlN-like在外施乙烯利（ethephon,ETH）3、6、12、24 h后的表达情况,最终明确SlN-like调控激素途径响应病毒侵染的机制。【结果】通过分子克隆与融合PCR技术,从番茄品种Micro-Tom中克隆获得全长3 444 bp 的SlN-like,上传至NCBI获得序列号MW792493。通过生物信息学分析发现SlN-like含有TIR、NB-ARC和NACHT结构域,并与马铃薯（Solanum tuberosum）N-like（AAP44394.1）亲缘关系最近。SlN-like在番茄各组织中均有表达,在茎中的表达量最高,其次是根、花,叶和果实中的表达量最低。TMV-GFP侵染番茄后第5、7天SlN-like的表达显著高于PBS处理,分别是PBS处理的1.6和2.2倍,并且TMV-GFP侵染会使SlN-like的表达持续升高。TRV载体介导沉默番茄的SlN-like,发现沉默78.3%的SlN-like不会影响番茄生长表型,但可促进TMV-GFP侵染;实时荧光定量PCR分析发现沉默植株中ERF1的表达显著降低,仅为对照组的12.5%;外施乙烯利处理番茄3 h后SlN-like表达量升高,并在12 h达到最高峰,是对照组的2.71倍,24 h后恢复正常。【结论】番茄SlN-like属NBS-LRR类抗病蛋白,其表达受TMV侵染诱导,沉默SlN-like促进TMV-GFP侵染,降低乙烯相关基因ERF1的表达,而外施乙烯利导致SlN-like的差异表达,揭示了SlN-like作为正调控因子可能影响乙烯途径介导的番茄抗病毒防御。

关键词: 番茄, SlN-like, 烟草花叶病毒, 基因表达, 乙烯

Abstract:

【Objective】As an important vegetable crop, tomato (Solanum lycopersicum) is endangered by various biological factors including pests, fungi, bacteria and viruses. The objective of this study is to clarify the antiviral function and mechanism of S. lycopersicum resistance gene SlN-like, and to provide a theoretical basis for the genetic breeding of antiviral S. lycopersicum and the targeted development of the antiviral agents. 【Method】The full length of SlN-like was obtained from the Solanaceae Genomics Network database and was divided into four segments, fusion PCR was used to amplify the full length of sequence. Bioinformatics was used to analyze the evolutionary relationship, protein characteristics, conserved domains, subcellular location and interaction relationship of SlN-like. Real-time fluorescent quantitative PCR was used to analyze the SlN-like expression in S. lycopersicum roots, stems, leaves, flowers and fruits and its response after tobacco mosaic virus (TMV) infection. S. lycopersicum endogenous SlN-like was silenced using tobacco rattle virus (TRV)-mediated gene silencing technology, and the silent plants were inoculated with TMV-GFP to clarify the influence of SlN-like on virus infection. The expressions of abscisic acid (ABA), jasmonic acid (JA) and ethylene (ET) hormone-related genes in silenced plants, and the expression of SlN-like after application of ethephon (ETH) for 3, 6, 12 and 24 h were analyzed by real-time fluorescence quantitative PCR to investigate the mechanism of SlN-like regulatory hormone pathway in response to virus infection. 【Result】Through molecular cloning and fusion PCR technology, a 3 444 bp SlN-like was cloned from S. lycopersicum variety Micro-Tom, and uploaded to NCBI to obtain the sequence number MW792493. Through bioinformatics analysis, it was found that SlN-like contains TIR, NB-ARC and NACHT domains, and is closely related to Solanum tuberosum N-like (AAP44394.1). SlN-like expressed in all tissues of S. lycopersicum, with the highest expression in stems, followed by roots, flowers, leaves and fruits. After TMV-GFP infection S. lycopersicum at 5th and 7th day, the SlN-like expression level was higher than that of PBS treatment, and TMV-GFP infection would cause the expression of SlN-like to increase continuously. TRV vector induced silencing of SlN-like in S. lycopersicum, and it was found that silencing 78.3% of SlN-like did not affect tomato growth phenotype, but silencing SlN-like promoted the infection of TMV-GFP. Real-time fluorescent quantitative PCR analysis found that the expression of ERF1 in SlN-like-silent plants was significantly reduced, only 12.5% of that in the control group. The expression of SlN-like increased after 3 h of external application of ethephon, and reached the highest peak at 12 h, which was 2.71 times that of the control group, and returned to normal at 24 h. 【Conclusion】S. lycopersicum SlN-like belongs to the NBS-LRR disease-resistant protein family, its expression is induced by TMV infection. Silencing SlN-like can promote TMV-GFP infection and reduce the expression of ethylene-related gene ERF1, while external application of ethephon resulted in the differential expression of SlN-like, revealing that SlN-like participates in S. lycopersicum antiviral defense through the ethylene pathway.

Key words: Solanum lycopersicum, SlN-like, tobacco mosaic virus (TMV), gene expression, ethylene

刘昌云,李欣羽,田绍锐,王靖,裴悦宏,马小舟,樊光进,汪代斌,孙现超. 番茄SlN-like的克隆、表达与抗病毒功能[J]. 中国农业科学, 2021, 54(20): 4348-4357.

LIU ChangYun,LI XinYu,TIAN ShaoRui,WANG Jing,PEI YueHong,MA XiaoZhou,FAN GuangJin,WANG DaiBin,SUN XianChao. Cloning, Expression and Anti-Virus Function Analysis of Solanum lycopersicum SlN-like[J]. Scientia Agricultura Sinica, 2021, 54(20): 4348-4357.

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引物名称 Primer name		引物序列F Primer sequence F (5′-3′)	引物序列R Primer sequence R (5′-3′)
PCR	P1	ATGAATCAGGAAAGTTCAGTGC	CGGCTAATGCATCTAACTGTTC
	P2	GAACAGTTAGATGCATTAGCCG	CATGCCAATCAAGCCACCTC
	P3	TGGCACTGACAAGATCGAAG	CATCGAGAATCAATCCCTCC
	P4	GGAGGGATTGATTCTCGATG	TCAATTCTCATACACAAGACG
qPCR	SlN-like	ACTTTCCTAGCCCACGTACC	ATACACAAGACGCACTCCA
	TMV MP	TTAGGTTCCCTGACGGTGAC	ACCGTTGCGTCGTCTACTCT
	ABI1	GCCGTTGTTTGTTCATCTC	ACTGTATCACCTTGCCTCC
	ERF1	GCTCTTAACGTCGGATGGTC	AGCCAAACCCTAGCTCCATT
	COI1	CAGCAGCCCATTGTTTCTTAC	TACTGGCCAAGTACTTCCAATC
	ACTIN	CGGTGCCCACTTTCCGATCT	TCCTCACCGTCAGCCATTTT
VIGS	TRV:SlN-like	CCGGAATTCCAATTCCGTCCTCTATGTCTC	CCGCTCGAGGTTACCATCGAGAATCAATC

番茄SlN-like的克隆、表达与抗病毒功能

Cloning, Expression and Anti-Virus Function Analysis of Solanum lycopersicum SlN-like

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