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植物研究 ›› 2021, Vol. 41 ›› Issue (4): 522-530.doi: 10.7525/j.issn.1673-5102.2021.04.007

• 研究报告 • 上一篇    

丹参体内Sm-miR858对R2R3-MYB转录因子SmPAP1进行靶向负调控作用研究

陈芳1, 佘婷婷1, 张琳1, 高浩天2, 李国梁2, 王健1,2()   

  1. 1.安康学院现代农业与生物科技学院,安康 725000
    2.陕西科技大学食品与生物工程学院,西安 710021
    3.教育部药用植物资源与天然药物化学重点实验室和西北濒危药材资源开发国家工程实验室,陕西师范大学生命科学学院,西安 710062
  • 收稿日期:2020-05-07 出版日期:2021-07-20 发布日期:2021-03-24
  • 通讯作者: 王健 E-mail:wangjianswj@163.com
  • 作者简介:陈芳(1979—),女,博士,副教授,主要从事基因功能与分子调控研究。
  • 基金资助:
    国家自然科学基金(31170281);陕西省科技厅项目(2019JQ-560);陕西省教育厅专项基金(16JK1014);安康学院科技创新项目(2017AYQDZR05)

Mechanism of Sm-miR858 Negatively Regulateda R2R3-MYB Transcription Factor SmPAP1 in Salvia miltiorrhiza

Fang CHEN1, Ting-Ting SHE1, Lin ZHANG1, Hao-Tian GAO2, Guo-Liang LI2, Jian WANG1,2()   

  1. 1.School Modern Agriculture and Biotechnology,Ankang University,Ankang 725000
    2.School of Food and Biological Engineering,Shaanxi University of Science and Technology,Xi’an 710021
    3.Key Laboratory of Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry,National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China,College of Life Sciences,Shaanxi Normal University,Xi’an 710062
  • Received:2020-05-07 Online:2021-07-20 Published:2021-03-24
  • Contact: Jian WANG E-mail:wangjianswj@163.com
  • About author:CHEN Fang(1979—),female,Ph.D,associate professor,engaged in gene function and molecular regulation.
  • Supported by:
    National Natural Science Foundation of China(31170281);Science and technology department of Shaanxi province(2019JQ-560);Natural science foundation of Education department of Shaanxi province(16JK1014);Ankang University Research Foundatio(2017AYQDZR05)

摘要:

MicroRNAs(miRNAs)是一类对基因表达进行负调控的非编码小分子RNA。通过前期对丹参miRNAs的高通量测序得到了一个miR858成熟序列,命名为Sm-miR858。序列比对显示,Sm-miR858与其它植物中已鉴定的miR858序列高度保守;Small RNA Northern blotting结果显示Sm-miR858在丹参根、茎和叶组织中均有表达,叶中表达水平相对较高。为了探究Sm-miR858在丹参体内的功能,首先利用在线生物软件对Sm-miR858的靶标基因进行预测,psRNATarget分析结果显示,Sm-miR858的潜在靶标基因共有13个,其中一个靶标基因SmPAP1作为一个重要的转录因子参与丹参酚酸类活性物质的代谢调控。为了验证Sm-miR858对SmPAP1的靶向作用,采用Real-time quantitative PCR依次对烟草瞬时表达体系和丹参组织器官中的Sm-miR858与SmPAP1之间共表达相关性进行分析与实验验证。Real-time qPCR结果显示,在丹参组织中SmPAP1与Sm-miR858共表达水平存在显著的负相关性。进而分别构建Sm-miR858和SmPAP1过表达植物载体,并在烟草叶片中进行瞬时共表达研究。结果显示,与对照相比,Sm-miR858 过表达会导致SmPAP1的mRNA水平显著下降,说明在丹参体内Sm-miR858的确对SmPAP1基因表达进行靶向负调控。研究结果为阐明Sm-miR858在丹参体内酚酸类活性物质代谢途径调控作用奠定坚实的基础。

关键词: Sm-miR858, SmPAP1, 丹参, 靶标基因

Abstract:

MicroRNAs(miRNAs) are a class of non-coding small RNAs that negatively regulate gene expression. Through high-throughput sequencing miRNAsof Salvia miltiorrhiza, a mature sequence miR858 named Sm-miR858 was obtained. The sequence alignment showed that the Sm-miR858 was highly conserved with the identified miR858 sequences in other plants, and the result of Small RNA northern blotting showed that the Sm-miR858 was expressed in the root, stem and leaf tissues of S. miltiorrhiza, especially relatively high in leaves. To explore the function of Sm-miR858 in S. miltiorrhiza, the Sm-miR858 was predicted first online using biological software. The result of psRNATarget analysis showed that there were 13 potential target genes of Sm-miR858. One was SmPAP1, an important transcription factor, participated in the metabolic regulation on phenolic acid active substances of S. miltiorrhiza. To further verify the targeting effect of Sm-miR858 on SmPAP1, the correlation of co-expression between Sm-miR858 and SmPAP1 was analyzed and verified by Real-time qPCR in the tobacco transient expression system and S. miltiorrhiza tissues and organs. The Real-time qPCR results showed that there was a significant negative correlation co-expression between SmPAP1 and Sm-miR858 in S. miltiorrhiza. Then the Sm-miR858 and SmPAP1 over-expressing plant vectors were constructed, and the transient co-expression of the Sm-miR858 and SmPAP1 was subsequently assayed in tobacco. The results showed that the sm-mir858 overexpression resulted in a significant decrease of the SmPAP1 mRNA expression level, compared with the control, and this indicated that sm-mir858 did actually target and negatively regulate the expression of SmPAP1 in S. miltiorrhiza. Above results laid a solid foundation for elucidating the role of sm-mir858 in the regulation phenolic acids biosynthesis in S. miltiorrhiza.

Key words: Sm-miR858, SmPAP1, Salvia miltiorrhiza, Target gene

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