Sly-miR166b及其靶基因在番茄抗晚疫病中的作用初探

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

摘要/Abstract

摘要：

为探明sly-miR166b在番茄抗晚疫病中的作用,分别构建了sly-miR166b的过表达载体和沉默载体,将它们在番茄离体叶片中瞬时表达,结果发现,过表达sly-miR166b下调了其靶基因的表达量,与空载体对照相比,晚疫病菌接种后叶片上相对病斑面积明显变小;而沉默sly-miR166b则上调了其靶基因的表达量,病菌接种后叶片上相对病斑面积较对照明显变大。初步表明,过表达sly-miR166b增强了植株对晚疫病的抗性,沉默sly-miR166b增强了植株的感病性,这为全面揭示sly-miR166b的抗病机制奠定了基础。

关键词: 番茄, 晚疫病, miRNA, 靶基因, 抗病

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

中图分类号:

S641.2

张晓艺, 洪雨慧, 张媛媛, 栾雨时. Sly-miR166b及其靶基因在番茄抗晚疫病中的作用初探[J]. 园艺学报, 2021, 48(8): 1595-1604.

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.

图/表 9

表1 Sly-miR166b及STTM-miR166b的核苷酸序列

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

表2 qRT-PCR引物及序列

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

图1 番茄叶片Sly-miR166b在晚疫病菌侵染后的表达模式不同小写字母代表差异显著（P﹤0.05）。

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

图2 番茄叶片Sly-miR166b靶基因在晚疫病菌侵染后的表达模式不同小写字母代表差异显著（P < 0.05）。下同。

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.

图3 番茄基因组DNA（A）及sly-miR166b 前体（B）PCR扩增产物

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

图4 OE166b载体骨架（A）及质粒PCR扩增pre-sly-miR166b产物（B）

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

图5 Sly-miR166b的STTM骨架（A）与重组表达载体STTM-miR166b（B）结构示意图及质粒PCR扩增STTM-miR166b产物（C）

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）

图6 Sly-miR166b瞬时过表达、沉默以及空载体番茄叶片中sly-miR166b及其靶基因的表达

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

图7 sly-miR166b过表达（TO）、沉默（TS）以及空载体（EV）番茄叶片病斑及其相对病斑面积

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

参考文献 25

[1]	Bloch D, Puli M R, Mosquna A, Yalovsky S. 2019. Abiotic stress modulates root patterning via ABA-regulated microRNA expression in the endodermis initials. Development, 146 (17):dev177097.
[2]	Candar C B, Arican E, Zhang B L. 2016. Small RNA and degradome deep sequencing reveals drought-and tissue-specific microRNAs and their important roles in drought-sensitive and drought-tolerant tomato genotypes. Plant Biotechnol J, 14 (8):1727-1746. doi: 10.1111/pbi.12533 pmid: 26857916
[3]	Chen L, Meng J, He X L, Zhang M, Luan Y S. 2019. Solanum lycopersicum microRNA1916 targets multiple target genes and negatively regulates the immune response in tomato. Plant Cell Environ, 42 (4):1393-1407. doi: 10.1111/pce.13468
[4]	Feng L, Xia R, Liu Y L. 2019. Comprehensive characterization of miRNA and PHAS Loci in the diploid strawberry(Fragaria vesca)genome. Horticultural Plant Journal, 5 (6):255-267. doi: 10.1016/j.hpj.2019.11.004
[5]	Fry W E, Birch P R, Judelson H S, Grunwald N J, Danies G, Everts K L, Gevens A J, Gugino B K, Johnson D A, Johnson S B, McGrath M T, Myers K L, Ristaino J B, Roberts P D, Secor G, Smart C D. 2015. Five reasons to consider Phytophthora infestans a reemerging pathogen. Phytopathology, 105 (7):966-981. doi: 10.1094/PHYTO-01-15-0005-FI pmid: 25760519
[6]	Hu G, Fan J, Xian Z, Huang W, Lin D, Li Z. 2014. Overexpression of SlREV alters the development of the flower pedicel abscission zone and fruit formation in tomato. Plant Sci, 229:86-95. doi: 10.1016/j.plantsci.2014.08.010 URL
[7]	Jiang N, Meng J, Cui J, Sun G X, Luan Y S. 2018. Function identification of miR482b,a negative regulator during tomato resistance to Phytophthora infestans. Hortic Res, 5:9. doi: 10.1038/s41438-018-0017-2 URL
[8]	Kim J, Jung J H, Reyes J L, Kim Y S, Kim S Y, Chung K S, Kim J A, Lee M, Lee Y, Narry K V, Chua N H, Park C M. 2005. microRNA-directed cleavage of ATHB15 mRNA regulates vascular development in Arabidopsis inflorescence stems. Plant J, 42 (1):84-94. doi: 10.1111/tpj.2005.42.issue-1 URL
[9]	Lin Yu-ling, Zhang Qing-lin, Zeng You-jing, Chen Xiao-hui, Zhang Zi-hao, Chen Yu-kun, Lai Zhong-xiong. 2017. Analysis on evolutionary characteristics and the temporal and spatial expression patterns of miR166 gene family in Dimocarpus longan. Acta Horticulturae Sinica, 44 (12):2285-2295. (in Chinese)
	林玉玲, 张清林, 曾友竞, 陈晓慧, 张梓浩, 陈裕坤, 赖钟雄. 2017. 龙眼miR166家族的分子进化特性及时空表达分析. 园艺学报, 44 (12):2285-2295.
[10]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-∆∆Ct method. Methods, 25 (4):402-408. pmid: 11846609
[11]	Luan Y S, Cui J, Zhai J M, Li J, Han L, Meng J. 2015. High-throughput sequencing reveals differential expression of miRNAs in tomato inoculated with Phytophthora infestans. Planta, 241 (6):1405-1416. doi: 10.1007/s00425-015-2267-7 URL
[12]	Malinovsky F G, Fangel J U, Willats W G. 2014. The role of the cell wall in plant immunity. Front Plant Sci, 5:178.
[13]	McConnell J R, Emery J, Eshed Y, Bao N, Bowman J, Barton M K. 2001. Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature, 411 (6838):709-713. doi: 10.1038/35079635 URL
[14]	Ohashi I K, Kubo M, Demura T, Fukuda H. 2005. Class Ⅲ homeodomain leucine-zipper proteins regulate xylem cell differentiation. Plant Cell Physiol, 46 (10):1646-1656. pmid: 16081527
[15]	Prigge M J, Otsuga D, Alonso J M, Ecker J R, Drews G N, Clark S E. 2005. Class Ⅲ homeodomain-leucine zipper gene family members have overlapping,antagonistic,and distinct roles in Arabidopsis development. Plant Cell, 17 (1):61-76. doi: 10.1105/tpc.104.026161 URL
[16]	Rodriguez R E, Mecchia M A, Debernardi J M, Schommer C, Weigel D, Palatnik J F. 2010. Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development, 137 (1):103-112. doi: 10.1242/dev.043067 pmid: 20023165
[17]	Salvador G R, Hsing Y I, San S B. 2018. The Polycistronic miR166k-166h positively regulates rice immunity via post-transcriptional control of EIN2. Front Plant Sci, 9:337. doi: 10.3389/fpls.2018.00337 pmid: 29616057
[18]	Su Y Y, Li H G, Wang Y L, Li S, Wang H L, Yu L, He F, Yang Y L, Feng C H, Shuai P, Liu C, Yin W L, Xia X L. 2018. Poplar miR472a targeting NBS-LRRs is involved in effective defence against the necrotrophic fungus Cytospora chrysosperma. J Exp Bot, 69 (22):5519-5530.
[19]	Valiollahi E, Farsi M, Kakhki A M. 2014. Sly-miR166 and Sly-miR319 are components of the cold stress response in Solanum lycopersicum. Plant Biotechnol Rep, 8:349-356. doi: 10.1007/s11816-014-0326-3 URL
[20]	Wang K T, Su X M, Cui X, Du Y C, Zhang S B, Gao J C. 2018. Identification and characterization of microRNA during Bemisia tabaci infestations in Solanum lycopersicum and Solanum habrochaites. Horticultural Plant Journal, 4 (2):62-72. doi: 10.1016/j.hpj.2018.03.002 URL
[21]	Wang J Y, Yu W G, Yang Y W, Li X, Chen T Z, Liu T L, Ma N, Yang X, Liu R Y, Zhang B L. 2015. Genome-wide analysis of tomato long non-coding RNAs and identification as endogenous target mimic for microRNA in response to TYLCV infection. Sci Rep, 5:16946. doi: 10.1038/srep16946 URL
[22]	Wang Xiao, Liu Yang, Chen Hai-yin, Liu Kun, Qi Ming-fang, Li Tian-lai, Liu Yu-feng. 2016. The effect of low night temperature on miRNA expression in mutant and wild type tomato leaf. Acta Horticulturae Sinica, 43 (12):2369-2379. (in Chinese)
	王孝, 刘阳, 陈海银, 刘琨, 齐明芳, 李天来, 刘玉凤. 2016. 低夜温下番茄 ABA 缺失突变体和野生型叶片中 miRNA 差异表达分析. 园艺学报, 43 (12):2369-2379.
[23]	Wu Liangliang, Zhang Ning, Zhu Xi, Ma Rui, Tang Xun, Yang Jiangwei, Si Huaijun. 2019. Identification and expression analysis of StmiR166b and its target genes in potato. Acta Horticulturae Sinica, 46 (7):1333-1343. (in Chinese)
	武亮亮, 张宁, 朱熙, 马瑞, 唐勋, 杨江伟, 司怀军. 2019. 马铃薯 StmiR166b 靶基因的鉴定及其表达分析. 园艺学报, 46 (7):1333-1343.
[24]	Yan J, Gu Y Y, Jia X Y, Kang W J, Pan S J, Tang X Q, Chen X M, Tang G L. 2012. Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell, 24 (2):415-427. doi: 10.1105/tpc.111.094144 URL
[25]	Zhang T, Zhao Y L, Zhao J H, Wang S, Jin Y, Chen Z Q, Fang Y Y, Hua C L, Ding S W, Guo H S. 2016. Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nature Plants, 2 (10):16153. doi: 10.1038/nplants.2016.153 pmid: 27668926