生物技术通报 ›› 2021, Vol. 37 ›› Issue (3): 18-26.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0917

• 研究报告 • 上一篇    下一篇

柽柳ThWRKY4转录因子结合ARR1AT元件调控基因表达

徐红云1(), 张恩辉1, 于存2   

  1. 1.贵州民族大学生态环境工程学院,贵阳 550025
    2.贵州大学林学院,贵阳 550025
  • 收稿日期:2020-07-22 出版日期:2021-03-26 发布日期:2021-04-02
  • 作者简介:徐红云,女,博士,研究方向:植物抗逆生理及分子生物学;E-mail:xhyplant@126.com
  • 基金资助:
    贵州民族大学自然科学类基金项目(0703001018002032);贵州民族大学创新训练项目(2018520236)

Tamarix hispida Transcription Factor ThWRKY4 Binds to ARR1AT Motif to Regulate Gene Expression

XU Hong-yun1(), ZHANG En-hui1, Yu Cun2   

  1. 1. College of Eco-Environment Engineering,Guizhou Minzu University,Guiyang 550025
    2. College of Forestry,Guizhou University,Guiyang 550025
  • Received:2020-07-22 Published:2021-03-26 Online:2021-04-02

摘要:

筛选和鉴定ThWRKY4能够特异性结合的顺式作用元件,并研究其对下游基因的表达调控,为了解WRKY转录因子调控基因表达的作用机制奠定基础。利用酵母反式单杂交、酵母单杂交、烟草瞬时转化和染色体免疫共沉淀技术分析ThWRKY4蛋白对ARR1AT元件的结合特性及对基因的表达调控情况。结果表明,ThWRKY4能够结合一个新的顺式作用元件ARR1AT(AATCG);ThWRKY4能够特异性结合ARR1AT元件,而不能结合ARR1AT的突变元件AM1-AM5(CCTCG、AACCG、AATAG、AATCA、CCCAA);ThWRKY4能够特异性结合含有ARR1AT元件的启动子片段而驱动报告基因的表达,而未能结合缺失ARR1AT元件的启动子片段。ThWRKY4能够于植体内结合ARR1AT元件调控下游基因的表达。

关键词: 柽柳, ThWRKY4, 酵母单杂交, 顺式作用元件, ChIP, 基因表达调控

Abstract:

The aim of this study is to screen and identify the cis-acting element specifically binding with Tamarix hispida transcription factor ThWRKY4,and to study the expression of downstream genes regulated by ThWRKY4,for laying a foundation in studying the mechanism of WRKY transcription factors regulating gene expression. We employed the transcription factor-centered yeast one-hybrid assay,yeast one-hybrid,tobacco transient transformation,and chromatin immunoprecipitation coupled with quantitative PCR(qChIP-PCR)to study the specificity of ThWRKY4 binding to ARR1AT and its regulation to gene expression. We identified a cis-acting element in ARR1AT(AATCG)bound by ThWRKY4. ThWRKT4 specifically bound to this ARR1AT element,but failed to bind to mutants(CCTCG,AACCG,AATAG,AATCA,and CCCAA). ThWRKY4 specifically bound to truncated promoters containing the ARR1AT motif,thus driving the expression of report gene,while not to the same truncated promoter lacking the ARR1AT motif. In conclusion,ThWRKT4 could regulate the expression of downstream genes by binding the ARR1AT element in vivo plants.

Key words: Tamarix hispida, ThWRKY4, yeast one-hybrid, cis-acting element, ChIP, gene expression and regulation