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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (11): 23-31    DOI: 10.13523/j.cb.2107013
研究报告     
sly-miR399在番茄抗晚疫病中的初步探究*
杨茜,栾雨时()
大连理工大学生物工程学院 大连 116024
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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摘要：

背景: 番茄(Solanum lycopersicum)是广泛种植的最具价值的果蔬之一,番茄晚疫病会导致其产量和品质降低。microRNAs(miRNAs)是一类内源性非编码RNA,广泛参与基因转录后调控。已有研究表明miR399家族可参与调控植物抗病过程。目的:探究番茄miR399(sly-miR399)对番茄抗晚疫病的影响。方法:构建sly-miR399的过表达和沉默载体,利用农杆菌介导在番茄叶片中瞬时表达;实时荧光定量PCR(qRT-PCR)技术检测相关基因表达水平;台盼蓝染色并统计分析瞬时过表达和瞬时沉默sly-miR399的番茄叶片的病斑情况。结果:瞬时过表达sly-miR399导致其靶基因UBC24的表达量下降了75%,病程相关蛋白(pathogenesis related proteins,PRs)SlPR1、SlPR2、SlPR3和SlPR5的表达量分别上升了3.6倍、2.2倍、2.3倍和6.4倍,茉莉酸(JA)信号通路相关基因SlJA1、SlLOX1和SlLOX2的表达量分别上升了1.3倍、2.5倍和1.5倍,JA转录抑制因子SlJAZ1的表达量下降了50%,接种晚疫病菌后,叶片上相对病斑面积明显减小;瞬时沉默导致其靶基因的表达量上升了1.8倍,PRs基因的表达量分别下降了65%、82%、52%和80%,SlJA1、SlLOX1和SlLOX2的表达量分别下降了84%、50%和65%,SlJAZ1的表达量上升了1.8倍,病菌接种后叶片上相对病斑面积明显变大。结论:初步揭示了sly-miR399在番茄抗晚疫病过程中的正调控因子作用。
关键词： 番茄;  晚疫病;  miR399;  瞬时表达    
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
收稿日期: 2021-07-04 出版日期: 2021-12-01
ZTFLH:  Q812  
基金资助: * 国家自然科学基金(31872116)
通讯作者: 栾雨时     E-mail: luanyush@dlut.edu.cn
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杨茜
栾雨时

引用本文:

杨茜,栾雨时. sly-miR399在番茄抗晚疫病中的初步探究*[J]. 中国生物工程杂志, 2021, 41(11): 23-31.

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

链接本文:

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

引物名称 正向引物序列(5'-3') 反向引物序列(5'-3')
sly-miR399 TTGAAAAGTGAAGGTCCTTG TTTACATATTCCATTTGGTG
UBC24 TTCTCCGCACAACCCTTCTA AATACCAATACTGCCAACGA
ARF17 AAAACTGCCTAATGGGCTTCT ATGGGCTACTGAGTCCAATCGT
F-box CTTTATTGGGCTTTATCTGC GTCCACGACACTTCTACTCC
NB-ARC TGGAGGAGGATGAGTCTTGG TTTGTTATGAGGCTCGGACA
SlPR1 CCAAGACTATCTTGCGGTTCA CGCTCTTGAGTTGGCATAGT
SlPR2 TCCAGGTAGAGACAGTGGTAAA CCTAAATATGTCGCGGTTGAGA
SlPR3 GAACGAGCTGGACAAGGTATT CGTTGTGGCATGATGGTTTATT
SlPR5 CCCAAACACCCTAGCTGAAT GGGCGAAAGTCATCGGTATATTA
SlJA1 GTGAGATCCGTGGAAAAAGAAGG CTTTTCTTCTCCCCAGCTTGT
SlJAZ1 GGAAAATCAAATGCCTAACCAG GGTACTTTTGCAGTTAGCCTA
SlLOX1 GTTGGACATGGTGACAAGAAAG GGCCAGAAGTTACCCAAACTA
SlLOX2 GATATAAGTGCGGAGAGTCGTG AAGCCTGGAGGTTGAGAATG
Actin TGTGTTGGACTCTGGTGATGGTGT ATCCAAACGAAGAATGGCATGCGG
表1  qRT-PCR引物及序列
图1  sly-miR399及其靶基因在晚疫病菌侵染后的相对表达量
图2  sly-miR399前体PCR扩增产物电泳
图3  sly-miR399过表达载体
图4  sly-miR399沉默载体
图5  瞬时过表达及沉默sly-miR399番茄叶片中sly-miR399(a)及其靶基因(b)的表达
图6  瞬时过表达及沉默sly-miR399番茄叶片中PRs基因的相对表达量
图7  瞬时过表达及沉默sly-miR399番茄叶片中茉莉酸信号通路相关基因的相对表达量
图8  瞬时过表达及沉默sly-miR399番茄叶片上病斑表型和台盼蓝染色(a)及相对病斑面积(b)统计
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