生物技术通报 ›› 2021, Vol. 37 ›› Issue (3): 18-26.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0917
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:基金资助:
XU Hong-yun1(), ZHANG En-hui1, Yu Cun2
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。
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.
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 |
表1 引物序列
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 |
[1] |
Su T, Xu Q, Zhang FC, et al. WRKY42 modulates phosphate homeostasis through regulating phosphate translocation and acquisition in Arabidopsis[J]. Plant Physiology, 2015,167(4):1579-1591.
doi: 10.1104/pp.114.253799 URL |
[2] |
Gong X, Zhang J, Hu J, et al. FcWRKY70, a WRKY protein of Fortunella crassifolia, functions in drought tolerance and modulates putrescin synjournal by regulating arginine decarboxylase gene[J]. Plant, Cell and Environment, 2015,38(11):2248-2262.
URL pmid: 25808564 |
[3] |
Zhou L, Wang NN, Gong SY, et al. Overexpression of a cotton(Gossypium hirsutum)WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants[J]. Plant Physiology Biochemistry, 2015,96(1):311-320.
doi: 10.1016/j.plaphy.2015.08.016 URL |
[4] |
Hiroaki A, Takaaki N, Noriko M, et al. WRKY transcription factors phosphorylated by MAPK regulate a plant immune NADPH oxidase in Nicotiana benthamiana[J]. The Plant Cell, 2015,27(9):2645-2663.
URL pmid: 26373453 |
[5] | Cai M, Qiu D, Yuan T, et al. Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance[J]. Plant Cell Environment, 2008,31(1):86-96. |
[6] |
Choi C, Hwang SH, Fang IR, et al. Molecular characterization of Oryza sativa WRKY6, which binds to W-box-like element 1 of the Oryza sativa pathogenesis related(PR)10a promoter and confers reduced susceptibility to pathogens[J]. New Phytologist, 2015,208(3):846-859.
doi: 10.1111/nph.13516 URL |
[7] |
Sun C, Palmqvist S, Olsson H, et al. A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar responsive elements of the iso1 promoter[J]. The Plant Cell, 2003,15(9):2076-2092.
doi: 10.1105/tpc.014597 URL pmid: 12953112 |
[8] |
Grierson C, Du JS, Torres Zabala M, et al. Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene[J]. The Plant Journal, 1994,5(6):815-826.
doi: 10.1046/j.1365-313x.1994.5060815.x URL pmid: 8054988 |
[9] |
Mangelsen E, Kilian J, Berendzen KW, et al. Phylogenetic and comparative gene expression analysis of barley(Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions between monocots and dicots[J]. BMC Genomics, 2008,9(1):194-204.
doi: 10.1186/1471-2164-9-194 URL |
[10] |
Van Verk MC, Pappaioannou D, Neeleman L, et al. A novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors[J]. Plant Physiology, 2008,146(4):1983-1995.
doi: 10.1104/pp.107.112789 URL pmid: 18263781 |
[11] |
Machens F, Becker M, Umrath F, et al. Identifcation of a novel type of WRKY transcription factor binding site in elicitor-responsive cis-sequences from Arabidopsis thaliana[J]. Plant Molecular Biology, 2014,84(4):371-385.
doi: 10.1007/s11103-013-0136-y URL |
[12] |
Zheng L, Liu G, Meng X, et al. A WRKY gene from Tamarix hispida, ThWRKY4, mediates abiotic stress responses by modulating reactive oxygen species and expression of stress-responsive genes[J]. Plant Molecular Biology, 2013,82(4):303-320.
doi: 10.1007/s11103-013-0063-y URL |
[13] |
Ji X, Wang L, Nie X, et al. A novel method to identify the DNA motifs recognized by a defined transcription factor[J]. Plant Molecular Biology, 2014,86(4):367-380.
doi: 10.1007/s11103-014-0234-5 URL |
[14] |
Huang XS, Wang W, Zhang Q, et al. A basic helix-loop-helix transcription factor, PtrbHLH of Poncirus trifoliata confers cold tolerance and modulates peroxidase-mediated scavenging of hydrogen peroxide[J]. Plant Physiology, 2013,162(2):1178-1194.
doi: 10.1104/pp.112.210740 URL |
[15] |
Lu CA, Lin CC, Lee KW, et al. The SnRK1A protein kinase plays a key role in sugar signalling during germination and seedling growth of rice[J]. The Plant Cell, 2007,19(8):2484-2499.
URL pmid: 17766403 |
[16] | Haring M, Offermann S, Danker T, et al. Chromatin immunoprecipitation:optimization, quantitative analysis and data normalization[J]. Plant Methods, 2007,24(3):11-27. |
[17] |
Shi H, Ye T, Zhong B, et al. AtHAP5A modulates freezing stress resistance in Arabidopsis through binding to CCAAT motif of AtXTH21[J]. New Phytologist, 2014,203(2):554-567.
doi: 10.1111/nph.12812 URL |
[18] |
Taniguchi M, Sasaki N, Tsuge T, et al. ARR1 directly activates cytokinin response genes that encode proteins with diverse regulatory functions[J]. Plant Cell Physiology, 2007,48(2):263-277.
doi: 10.1093/pcp/pcl063 URL pmid: 17202182 |
[19] |
Oka A, Sakai H, Iwakoshi S. His-Asp phosphorelay signal transduction in higher plants:Receptors and response regulators for cytokinin signalling in Arabidopsis thaliana[J]. Genes Genetic Systems, 2002,77(6):383-391.
doi: 10.1266/ggs.77.383 URL pmid: 12589073 |
[20] |
Sakai H, Aoyama T, Oka A. Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators[J]. The Plant Journal, 2000,24(6):703-711.
doi: 10.1046/j.1365-313x.2000.00909.x URL pmid: 11135105 |
[21] |
Li J, Sima W, Ouyang B, et al. Identification and expression pattern of a ZPR1 gene in wild tomato(Solanum Pennellii)[J]. Plant Molecular Biology Reports, 2013,31(2):409-417.
doi: 10.1007/s11105-012-0509-4 URL |
[22] |
Yuan J, Chen D, Ren YJ, et al. Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice[J]. Plant Physiology, 2008,146(4):1637-1650.
URL pmid: 18258694 |
[23] |
Sharma N, Russell SD, Bhalla PL, et al. Putative cis-regulatory elements in genes highly expressed in rice sperm cells[J]. BMC Research Notes, 2011,4(319):319-409.
doi: 10.1186/1756-0500-4-319 URL |
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