Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (20): 4348-4357.doi: 10.3864/j.issn.0578-1752.2021.20.009

• PLANT PROTECTION • Previous Articles     Next Articles

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

LIU ChangYun1(),LI XinYu1,TIAN ShaoRui1,WANG Jing1,PEI YueHong1,MA XiaoZhou1,2,FAN GuangJin1,WANG DaiBin3(),SUN XianChao1()   

  1. 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 E-mail:15228920380@163.com;467572562@qq.com;sunxianchao@163.com

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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

Table 1

Primers used in this study"

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

Fig. 1

Phylogenetic analyses of SlN-like and its homologues"

Fig. 2

Expression analysis of SlN-like"

Fig. 3

Phenotype and silencing efficiency detection in SlN-like silenced plant"

Fig. 4

Silencing of SlN-like promotes TMV-GFP infection"

Fig. 5

Hormone-related gene expression in SlN-like silenced plant"

Fig. 6

SlN-like expression after external application of ethephon"

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