Preliminary Study on the Role of sly-miR166b and Its Target Genes in Tomato Resistance to Late Blight

doi:10.16420/j.issn.0513-353x.2020-0730

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (8): 1595-1604.doi: 10.16420/j.issn.0513-353x.2020-0730

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Preliminary Study on the Role of sly-miR166b and Its Target Genes in Tomato Resistance to Late Blight

ZHANG Xiaoyi, HONG Yuhui, ZHANG Yuanyuan, LUAN Yushi()

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

Received:2020-09-03 Revised:2021-03-07 Online:2021-08-25 Published:2021-09-06
Contact: LUAN Yushi E-mail:luanyush@dlut.edu.cn

Abstract

Abstract:

To investigate the function of sly-miR166b in tomato resistance to late blight,the vectors that overexpressing and silencing sly-miR166b were constructed and transiently expressed in tomato plants respectively. The results showed that transient overexpressing sly-miR166b（TO）reduced its target genes expression. The ratio of lesion area to leaf area on TO leaves was much lower than that in control leaves upon inoculation. On the contrary,transient silencing sly-miR166b（TS）increased its target genes expressions. The ratio of lesion area to leaf area on TS leaves was much higher than that in control leaves upon inoculation. This study preliminarily showed that overexpression of sly-miR166b enhanced plant resistance to late blight,while silencing sly-miR166b enhanced plant susceptibility to late blight,laying a foundation for comprehensively revealing the sly-miR166b mechanism in disease resistance.

Key words: tomato, late blight, miRNA, target gene, disease resistance

CLC Number:

S641.2

ZHANG Xiaoyi, HONG Yuhui, ZHANG Yuanyuan, LUAN Yushi. Preliminary Study on the Role of sly-miR166b and Its Target Genes in Tomato Resistance to Late Blight[J]. Acta Horticulturae Sinica, 2021, 48(8): 1595-1604.

Figures/Tables 9

Table 1 Nucleotide sequences of sly-miR166b and STTM-miR166b

序列名称 Sequence name	序列（5′-3′） Sequence
Sly-miR166b	UCGGACCAGGCUUCAUUCCCC
STTM-miR166b	CGGGATCCCGGGGAATGAAGCctaCTGGTCCGAGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATGGGGAATGAAGCctaCTGGTCCGACGAGCTCG

Table 2 Primer sequences for qRT-PCR

序列名称 Sequence name	正向引物（5′-3′） Forward sequence	反向引物（5′-3′） Reverse sequence
Sly-miR166b	TCGGACCAGGCTTCATTCCCC	Universal primers supplied in kit
SlHDZ1	CAATGGCCATAGCACCCTCT	TAGCTCCGGGAAATCCACCT
SlHDZ2	TTCCCGAAGTCCTCAGACCA	CCTCAAAACAGCAGGTTGCC
SlHDZ3	TGCTTCAATGGCTCGACAGT	TCCCCAATGGCAAACGAAGA
SlHDZ4	TCGCAGAGGACAACTATGGC	ACGGCCTCGTTAAAACCCTT
SlHDZ5	GCAGAAACAAGTCTCGCAGC	AGTCCAGCAGGGTTGTTAGC
SlHDZ6	CCTCGTGACTTCTGGCTGAT	TTGCGGCATACTTGGACCAT
Actin	ACCTTCAACGTTCCAGCTATG	TCACCAGAGTCCAACACAATA

Fig. 1 Expression pattern of sly-miR166b upon Phytophthora infestans infection Significant differences are presented with different lowercase letters（P < 0.05）.

Fig. 2 Expression pattern of sly-miR166b target genes upon Phytophthora infestans infection Significant differences are presented with different lowercase letters（P < 0.05）. The same below.

Fig. 3 The electrophoresis of tomato genome DNA（A） and the PCR product of pre-sly-miR166b（B）

Fig. 4 Schematic diagram of OE166b vector（A）and electrophoresis of PCR product of pre-sly-miR166b（B）

Fig. 5 Schematic diagram of sly-miR166b STTM vector（A）and recombinant expression vector STTM166b（B）,and electrophoresis of PCR product of STTM-miR166b（C）

Fig. 6 Expression levels of Sly-miR166b and its target genes in tomato leaves that transiently overexpressing sly-miR166b,transiently silencing sly-miR166b and empty vector

Fig. 7 Lesion symptoms and ratio of lesion area to leaf area on leaves of transiently overexpressing sly-miR166b（TO）,transiently silencing sly-miR166b（TS）and empty vector（EV）tomato plants

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Preliminary Study on the Role of sly-miR166b and Its Target Genes in Tomato Resistance to Late Blight

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