Preliminary Study of Sly-miR399 in Tomato Resistance to Late Blight

doi:10.13523/j.cb.2107013

China Biotechnology

2021, Vol. 41

Issue (11): 23-31 DOI: 10.13523/j.cb.2107013

Preliminary Study of Sly-miR399 in Tomato Resistance to Late Blight

YANG Xi,LUAN Yu-shi(

)

School of Bioengineering, Dalian University of Technology, Dalian 116024, China

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Abstract

Background: Tomato (Solanum lycopersicum) is one of the most valuable fruits and vegetables widely cultivated. Late blight disease of tomato reduces the quality and yield of tomatoes. MicroRNAs (miRNAs) area class of endogenous non-coding RNAs, which are widely involved in post-transcriptional regulation of genes. Recent evidence suggests that miR399 family participates in the regulation of plant disease resistance. Objective: Investigate the effect of sly-miR399 in tomato to late blight. Methods: The overexpressing and silencing vectors of sly-miR399 is constructed and transiently expressed in tomato leaves by the Agrobacterium-mediated method. Quantitative real-time PCR (qRT-PCR) is used to detect the related genes expression level. Trypan blue staining is used to analyze the lesion condition of tomato leaves with transient overexpressing (TO) and silencing (TS) sly-miR399. Results: Transient overexpressing sly-miR399 reduced the expression of its target gene UBC24 by 1.5 times and increased the expression of pathogenesis related proteins (PRs) SlPR1, SlPR2, SlPR3 and SlPR5 by 3.6 times, 2.2 times 2.3 times and 6.4 times, respectively. It increased the expression of JA related genes SlJA1, SlLOX1 and SlLOX2 by 1.3 times, 2.5 times and 1.5 times, respectively and reduced the expression of JA transcription repressor SlJAZ1 by 50%. After infection, the relative diseased area on TO leaves was reduced significantly. Transient silencing sly-miR399 increased the expression of its target gene by 1.8 times and reduced the expressions of PRs genes by 65%, 82%, 52% and 80%, and it decreased the expression of SlJA1, SlLOX1 and SlLOX2 by 84%, 50% and 65%,respectively and increased the expression of SlJAZ1 by 1.8 times. After infection, the relative diseased area on TS leaves increased significantly. Conclusion: sly-miR399 plays a positive regulatory role in tomato resistance to late blight.

Key words： Tomato Late blight miR399 Transient expression

Received: 04 July 2021 Published: 01 December 2021

ZTFLH:

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Corresponding Authors: Yu-shi LUAN E-mail: luanyush@dlut.edu.cn

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Cite this article:

YANG Xi,LUAN Yu-shi. Preliminary Study of Sly-miR399 in Tomato Resistance to Late Blight. China Biotechnology, 2021, 41(11): 23-31.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2107013 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I11/23

Table 1 Primer and its sequences for qRT-PCR

Fig.1 Relative expression of sly-miR399 and its target genes upon P. infestans infection Hpi represents for hours post infection;Significant differences (P<0.05) are presented with different lowercase letters

Fig.2 The PCR product of sly-pre-miR399 M: DL2000 DNA marker; 1: PCR product of sly-pre-miR399

Fig.3 sly-miR399 overexpressing vector (a)OE-399 vector structure (b)Electrophoresis of PCR product of sly-pre-miR399 M:DL2000 DNA marker; 1: PCR products of sly-pre-miR399 using plasmids as templates

Fig.4 sly-miR399 silencing vector (a)Schematic diagram of sly-miR399 STTM vector (b)STTM-miR399 vector structure (c)Electrophoresis of PCR product of STTM-miR399 M:DL2000 DNA marker; 1: PCR products of sly-pre-miR399 using plasmids as templates

Fig.5 Expression levels of sly-miR399(a)and its target genes(b)in tomato leaves That transiently overexpressing sly-miR399, transiently silencing sly-miR399. EV: Empty vector; TO399: Transiently overexpressing sly-miR399; TS399: Transiently silencing sly-miR399. Significant differences (P<0.05) are presented with different lowercase letters

Fig.6 Expression levels of PRs gene in tomato leaves that transiently overexpressing sly-miR399, transiently silencing sly-miR399 EV: Empty vector; TO399: Transiently overexpressing sly-miR399; TS399: Transiently silencing sly-miR399. Significant differences (P<0.05) are presented with different lowercase letters

Fig.7 Expression levels of JA related genes in tomato leaves that transiently overexpressing sly-miR399, transiently silencing sly-miR399 EV: Empty vector; TO399: Transiently overexpressing sly-miR399; TS399: Transiently silencing sly-miR399. Significant differences (P<0.05) are presented with different lowercase letters

Fig.8 Lesion symptoms and trypan blue staining (a) and ratio of lesion area to leaf area (b) on leaves of transiently overexpressing sly-miR399, transiently silencing sly-miR399 and empty vector EV: Empty vector; TO399: Transiently overexpressing sly-miR399; TS399: Transiently silencing sly-miR399. Significant differences (P<0.05) are presented with different lowercase letters


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