柽柳ThWRKY4转录因子结合ARR1AT元件调控基因表达

doi:10.13560/j.cnki.biotech.bull.1985.2020-0917

摘要/Abstract

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

徐红云, 张恩辉, 于存. 柽柳ThWRKY4转录因子结合ARR1AT元件调控基因表达[J]. 生物技术通报, 2021, 37(3): 18-26.

XU Hong-yun, ZHANG En-hui, Yu Cun. Tamarix hispida Transcription Factor ThWRKY4 Binds to ARR1AT Motif to Regulate Gene Expression[J]. Biotechnology Bulletin, 2021, 37(3): 18-26.

图/表 7

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用途	引物名称	序列（5'-3'）
酵母单杂交	PGADT7Rec2-ThWRKY4-F	AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCCATGGCAGTGGAATTAATGGTAG
	PGADT7Rec2-ThWRKY4-R	TCTAGAGGCCGAGGCGGCCGACATGCGATGATTCAAGCACGAGAGCAGCG
	pHIS2-ARR1AT-F	AATTC AATCG AATCG AATCG GAGCT
	pHIS2-ARR1AT-R	C CGATT CGATT CGATT G
	pHIS2-AM1-F	AATTC CCTCG CCTCG CCTCG GAGCT
	pHIS2-AM1-R	C CGAGG CGAGG CGAGG G
	pHIS2-AM2-F	AATTC AACCG AACCG AACCG GAGCT
	pHIS2-AM2-R	C CGGTT CGGTT CGGTT G
	pHIS2-AM3-F	AATTC AATAG AATAG AATAG GAGCT
	pHIS2-AM3-R	C CTATT CTATT CTATT G
	pHIS2-AM4-F	AATTC AATCA AATCA AATCA GAGCT
	pHIS2-AM4-R	C TGATT TGATT TGATT G
	pHIS2-AM5-F	AATTC CCCAA CCCAA CCCAA GAGCT
	pHIS2-AM5-R	C TTGGG TTGGG TTGGG G
	pHIS2-ARRp+ -F	CCGGAATTCCGATTTTAGTATTAACCGAAC
	pHIS2-ARRp+ -R	GACCGAGCTCTCTCAGTCTTCAGGCTAATTAG
	pHIS2-ARRp- -F	CCGGAATTCTTAGTATTAACCGAACCAATTATAG
	pHIS2-ARRp- -R	GACCGAGCTCTCTCAGTCTTCAGGCTAATTAG
烟草瞬时转化	pROK2-ThWRKY4-F	CGGGGGACTCTAGAGGATCCCCATGGCAGTGGAATTAATGGTAGGC
	pROK2-ThWRKY4-R	GGAAATTCGAGCTCGGTACCCCGATGATTCAAGCACGAGAGCA
	PCAMBIA1301-ARR1AT-F	GATCC AATCG AATCG AATCG A
	PCAMBIA1301-ARR1AT-R	AGCTT CGATT CGATT CGATT G
	PCAMBIA1301-AM5-F	GATCC CCCAA CCCAA CCCAA A
	PCAMBIA1301-AM5-R	AGCTT TTGGG TTGGG TTGGG G
	PCAMBIA1301-ARRp+ -F	CGCGGATCCCGATTTTAGTATTAACCGAAC
	PCAMBIA1301-ARRp+ -R	CCCAAGCTTTCTCAGTCTTCAGGCTAATTAG
	PCAMBIA1301-ARRp- -F	CGCGGATCCTTAGTATTAACCGAACCAATTATAG
qChIP-PCR	PCAMBIA1301-ARRp- -R	CCCAAGCTTTCTCAGTCTTCAGGCTAATTAG
	TUB-F	TCAATTTGAATCAAACTCATAG
	TUB-R	GCTCACTCCTCTCTGTGTTTG
	ARRp+ -F：	CGATTTTAGTATTAACCGAAC
	ARRp+ -R：	TCTCAGTCTTCAGGCTAATTAG

用途	引物名称	序列（5'-3'）
酵母单杂交	PGADT7Rec2-ThWRKY4-F	AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCCATGGCAGTGGAATTAATGGTAG
	PGADT7Rec2-ThWRKY4-R	TCTAGAGGCCGAGGCGGCCGACATGCGATGATTCAAGCACGAGAGCAGCG
	pHIS2-ARR1AT-F	AATTC AATCG AATCG AATCG GAGCT
	pHIS2-ARR1AT-R	C CGATT CGATT CGATT G
	pHIS2-AM1-F	AATTC CCTCG CCTCG CCTCG GAGCT
	pHIS2-AM1-R	C CGAGG CGAGG CGAGG G
	pHIS2-AM2-F	AATTC AACCG AACCG AACCG GAGCT
	pHIS2-AM2-R	C CGGTT CGGTT CGGTT G
	pHIS2-AM3-F	AATTC AATAG AATAG AATAG GAGCT
	pHIS2-AM3-R	C CTATT CTATT CTATT G
	pHIS2-AM4-F	AATTC AATCA AATCA AATCA GAGCT
	pHIS2-AM4-R	C TGATT TGATT TGATT G
	pHIS2-AM5-F	AATTC CCCAA CCCAA CCCAA GAGCT
	pHIS2-AM5-R	C TTGGG TTGGG TTGGG G
	pHIS2-ARRp+ -F	CCGGAATTCCGATTTTAGTATTAACCGAAC
	pHIS2-ARRp+ -R	GACCGAGCTCTCTCAGTCTTCAGGCTAATTAG
	pHIS2-ARRp- -F	CCGGAATTCTTAGTATTAACCGAACCAATTATAG
	pHIS2-ARRp- -R	GACCGAGCTCTCTCAGTCTTCAGGCTAATTAG
烟草瞬时转化	pROK2-ThWRKY4-F	CGGGGGACTCTAGAGGATCCCCATGGCAGTGGAATTAATGGTAGGC
	pROK2-ThWRKY4-R	GGAAATTCGAGCTCGGTACCCCGATGATTCAAGCACGAGAGCA
	PCAMBIA1301-ARR1AT-F	GATCC AATCG AATCG AATCG A
	PCAMBIA1301-ARR1AT-R	AGCTT CGATT CGATT CGATT G
	PCAMBIA1301-AM5-F	GATCC CCCAA CCCAA CCCAA A
	PCAMBIA1301-AM5-R	AGCTT TTGGG TTGGG TTGGG G
	PCAMBIA1301-ARRp+ -F	CGCGGATCCCGATTTTAGTATTAACCGAAC
	PCAMBIA1301-ARRp+ -R	CCCAAGCTTTCTCAGTCTTCAGGCTAATTAG
	PCAMBIA1301-ARRp- -F	CGCGGATCCTTAGTATTAACCGAACCAATTATAG
qChIP-PCR	PCAMBIA1301-ARRp- -R	CCCAAGCTTTCTCAGTCTTCAGGCTAATTAG
	TUB-F	TCAATTTGAATCAAACTCATAG
	TUB-R	GCTCACTCCTCTCTGTGTTTG
	ARRp+ -F：	CGATTTTAGTATTAACCGAAC
	ARRp+ -R：	TCTCAGTCTTCAGGCTAATTAG