Cloning of AcWRKY94 gene from kiwifruit and its functional analysis under salt stress

doi:10.3969/j.issn.1004-1524.20240485

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (11): 2501-2509.DOI: 10.3969/j.issn.1004-1524.20240485

• Horticultural Science • Previous Articles Next Articles

Cloning of AcWRKY94 gene from kiwifruit and its functional analysis under salt stress

GAO Jing¹^,²(), LU Linghong², GU Xianbin², FAN Fei², SONG Genhua², ZHANG Huiqin²^,^*()

1. College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
2. Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2024-06-05 Online:2024-11-25 Published:2024-11-27

Abstract

Abstract:

In order to clarify the function of AcWRKY94 under salt stress, the WRKY transcription factor AcWRKY94 was cloned from ‘Hongyang’ kiwifruit. Bioinformatics analysis of AcWRKY94 was carried out using relevant websites and biological software. The expression of AcWRKY94 under salt treatment was analyzed. AcWRKY94 was overexpressed in Nicotiana benthamiana by Agrobacterium-mediated transgenic technology, and the salt tolerance of transgenic tobacco plants were analyzed. The results showed that the open reading frame of the AcWRKY94 was 906 bp and encoded 301 amino acids. Promoter sequence analysis revealed that AcWRKY94 might respond to a variety of signals such as abscisic acid, salicylic acid, methyl jasmonate, gibberellin, drought, and anaerobic. Multiple sequence alignment and phylogenetic tree analysis showed that the homologous sequences of AcWRKY94 and several species contain a WRKYGQK conserved domain and a C₂HC zinc finger structure, which belong to WRKY Group III. Among them, the kiwifruit AcWRKY94 was closely related to the CsWRKY70-like proteins of tea plant. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of AcWRKY94 in roots of kiwifruit was significantly induced by salt treatment. After salt treatment, compared with wild type tobacco, the resistance of AcWRKY94-overexpressed line to salt was significantly enhanced, the content of malondialdehyde (MDA) and H₂O₂ decreased significantly, the content of proline (Pro) increased significantly, and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) increased significantly. The above results suggested that AcWRKY94 could be induced by salt stress and might be involved in the regulation of salt resistance in kiwifruit.

Key words: kiwifruit, WRKY, salt stress, function

CLC Number:

S663.4

GAO Jing, LU Linghong, GU Xianbin, FAN Fei, SONG Genhua, ZHANG Huiqin. Cloning of AcWRKY94 gene from kiwifruit and its functional analysis under salt stress[J]. Acta Agriculturae Zhejiangensis, 2024, 36(11): 2501-2509.

Figures/Tables 9

References 26

[1]	VAN ZELM E, ZHANG Y X, TESTERINK C. Salt tolerance mechanisms of plants[J]. Annual Review of Plant Biology, 2020, 71: 403-433.
[2]	LIANG X Y, LI J F, YANG Y Q, et al. Designing salt stress-resilient crops: current progress and future challenges[J]. Journal of Integrative Plant Biology, 2024, 66(3): 303-329.
[3]	MUNNS R. Genes and salt tolerance: bringing them together[J]. The New Phytologist, 2005, 167(3): 645-663.
[4]	MUNNS R, TESTER M. Mechanisms of salinity tolerance[J]. Annual Review of Plant Biology, 2008, 59: 651-681.
[5]	国庆. 小黑杨转录因子PsnHDZ01基因调控抗旱耐盐的分子机制研究[D]. 哈尔滨: 东北林业大学, 2022.
	GUO Q. Molecular mechanism of transcription factor PsnHDZ01 in regulation of drought and salt tolerance in Populus simonii×Populus nigra[D]. Harbin:Northeast Forestry University, 2022. (in Chinese with English abstract)
[6]	EULGEM T, RUSHTON P J, ROBATZEK S, et al. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science, 2000, 5(5): 199-206.
[7]	CHEN L G, ZHANG L P, LI D B, et al. WRKY8 transcription factor functions in the TMV-cg defense response by mediating both abscisic acid and ethylene signaling in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(21): E1963-E1971.
[8]	HU Y R, CHEN L G, WANG H P, et al. Arabidopsis transcription factor WRKY8 functions antagonistically with its interacting partner VQ9 to modulate salinity stress tolerance[J]. The Plant Journal, 2013, 74(5): 730-745.
[9]	QIU Y, YU D. Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis[J]. Environmental and Experimental Botany, 2009, 65(1): 35-47.
[10]	DONG Q L, ZHENG W Q, DUAN D Y, et al. MdWRKY30, a group IIa WRKY gene from apple, confers tolerance to salinity and osmotic stresses in transgenic apple callus and Arabidopsis seedlings[J]. Plant Science, 2020, 299: 110611.
[11]	相立, 赵蕾, 王玫, 等. 苹果MdWRKY74的克隆和功能分析[J]. 园艺学报, 2022, 49(3): 482-492.
	XIANG L, ZHAO L, WANG M, et al. Cloning and functional analysis of MdWRKY74 in apple[J]. Acta Horticulturae Sinica, 2022, 49(3): 482-492. (in Chinese with English abstract)
[12]	YU Y A, HE L Y, WU Y X. Wheat WRKY transcription factor TaWRKY24 confers drought and salt tolerance in transgenic plants[J]. Plant Physiology and Biochemistry, 2023, 205: 108137.
[13]	SHI W Y, DU Y T, MA J, et al. The WRKY transcription factor GmWRKY12 confers drought and salt tolerance in soybean[J]. International Journal of Molecular Sciences, 2018, 19(12): 4087.
[14]	ZHU H, JIANG Y N, GUO Y, et al. A novel salt inducible WRKY transcription factor gene, AhWRKY 75, confers salt tolerance in transgenic peanut[J]. Plant Physiology and Biochemistry, 2021, 160: 175-183.
[15]	LONG L X, GU L J, WANG S J, et al. Progress in the understanding of WRKY transcription factors in woody plants[J]. International Journal of Biological Macromolecules, 2023, 242(Pt 1): 124379.
[16]	WANG H P, CHEN W Q, XU Z Y, et al. Functions of WRKYs in plant growth and development[J]. Trends in Plant Science, 2023, 28(6): 630-645.
[17]	XING M Y, WANG W Q, ZHANG C, et al. Identification and functional analyses of the transcription factors AcWRKY117 and AcWRKY29 involved in waterlogging response in kiwifruit plant[J]. Scientia Horticulturae, 2024, 324: 112568.
[18]	GAN Z Y, YUAN X, SHAN N, et al. AcWRKY40 mediates ethylene biosynthesis during postharvest ripening in kiwifruit[J]. Plant Science, 2021, 309: 110948.
[19]	WANG J, LIU X F, ZHANG H Q, et al. Transcriptional and post-transcriptional regulation of ethylene biosynthesis by exogenous acetylsalicylic acid in kiwifruit[J]. Horticulture Research, 2022, 9: uhac116.
[20]	GULZAR F, FU J Y, ZHU C Y, et al. Maize WRKY transcription factor ZmWRKY79 positively regulates drought tolerance through elevating ABA biosynthesis[J]. International Journal of Molecular Sciences, 2021, 22(18): 10080.
[21]	DONG Q L, DUAN D Y, WANG F, et al. The MdVQ37-MdWRKY100 complex regulates salicylic acid content and MdRPM1 expression to modulate resistance to Glomerella leaf spot in apples[J]. Plant Biotechnology Journal, 2024, 22(8): 2364-2376.
[22]	WANG Z R, GAO M, LI Y F, et al. The transcription factor SlWRKY37 positively regulates jasmonic acid and dark-induced leaf senescence in tomato[J]. Journal of Experimental Botany, 2022, 73(18): 6207-6225.
[23]	JIANG J J, MA S H, YE N H, et al. WRKY transcription factors in plant responses to stresses[J]. Journal of Integrative Plant Biology, 2017, 59(2): 86-101.
[24]	LU K K, SONG R F, GUO J X, et al. CycC1;1-WRKY75 complex-mediated transcriptional regulation of SOS1 controls salt stress tolerance in Arabidopsis[J]. The Plant Cell, 2023, 35(7): 2570-2591.
[25]	MA J L, LI C H, SUN L L, et al. The SlWRKY57-SlVQ21/SlVQ16 module regulates salt stress in tomato[J]. Journal of Integrative Plant Biology, 2023, 65(11): 2437-2455.
[26]	YU J, ZHU C S, XUAN W, et al. Genome-wide association studies identify OsWRKY53 as a key regulator of salt tolerance in rice[J]. Nature Communications, 2023, 14(1): 3550.

引物名称 Primer name	正向引物序列(5'→3') Forward primer sequence (5'→3')	反向引物序列(5'→3') Reverse primer sequence (5'→3')	用途 Application
AcWRKY94-CDS	CAGTGGTCTCACAACATGGGCATCCTTCGGCCTGA	CAGTGGTCTCATACACTCACCCTCACCAAAGCAAA	基因克隆 Gene cloning
AcWRKY94-qPCR	AAGACCTTGGACGCATGGAT	ATTTGGTGGGGCTCCTCTTG	实时荧光定量PCR qRT-PCR
AcActin	TGGAATGGAAGCTGCAGGA	CACCACTGAGCACAATGTTGC	内参基因Reference gene
AcWRKY94-check	GAAGTGTGCGTGTGATTCGTGT	TGAGATTTTGAGGGTGTTTGTG	转基因苗鉴定
			Identification of transgenic seedling

引物名称 Primer name	正向引物序列(5'→3') Forward primer sequence (5'→3')	反向引物序列(5'→3') Reverse primer sequence (5'→3')	用途 Application
AcWRKY94-CDS	CAGTGGTCTCACAACATGGGCATCCTTCGGCCTGA	CAGTGGTCTCATACACTCACCCTCACCAAAGCAAA	基因克隆 Gene cloning
AcWRKY94-qPCR	AAGACCTTGGACGCATGGAT	ATTTGGTGGGGCTCCTCTTG	实时荧光定量PCR qRT-PCR
AcActin	TGGAATGGAAGCTGCAGGA	CACCACTGAGCACAATGTTGC	内参基因Reference gene
AcWRKY94-check	GAAGTGTGCGTGTGATTCGTGT	TGAGATTTTGAGGGTGTTTGTG	转基因苗鉴定
			Identification of transgenic seedling

作用元件 Functional element	序列 Sequence	位置 Position	位点功能 Function of site
ABRE	ACGTG	-160、-430、-734、-1005、-1613	参与ABA响应
			Cis-acting element involved in the abscisic acid responsiveness
TCA-element	CCATCTTTTT	-1170	参与SA响应
			Cis-acting element involved in salicylic acid responsiveness
CGTCA-motif	CGTCA	-171、-174、-185	参与MeJA响应
			Cis-acting regulatory element involved in the MeJA-responsiveness
GARE-motif	TCTGTTG	-463	参与赤霉素响应
			Cis-acting element involved in gibberellin-responsiveness
TATC-box	TATCCCA	-490、-1293、-1471	参与赤霉素响应
			Cis-acting element involved in gibberellin-responsiveness
MBS	CAACTG	-526	参与干旱诱导的MYB结合位点
			MYB binding site involved in drought-inducibility
ARE	AAACCA	-790、-1550	参与厌氧诱导
			Cis-acting regulatory element essential for the anaerobic induction
W box	TTGACC	-382	WRKY转录因子结合位点
			WRKY transcription factor binding site

作用元件 Functional element	序列 Sequence	位置 Position	位点功能 Function of site
ABRE	ACGTG	-160、-430、-734、-1005、-1613	参与ABA响应
			Cis-acting element involved in the abscisic acid responsiveness
TCA-element	CCATCTTTTT	-1170	参与SA响应
			Cis-acting element involved in salicylic acid responsiveness
CGTCA-motif	CGTCA	-171、-174、-185	参与MeJA响应
			Cis-acting regulatory element involved in the MeJA-responsiveness
GARE-motif	TCTGTTG	-463	参与赤霉素响应
			Cis-acting element involved in gibberellin-responsiveness
TATC-box	TATCCCA	-490、-1293、-1471	参与赤霉素响应
			Cis-acting element involved in gibberellin-responsiveness
MBS	CAACTG	-526	参与干旱诱导的MYB结合位点
			MYB binding site involved in drought-inducibility
ARE	AAACCA	-790、-1550	参与厌氧诱导
			Cis-acting regulatory element essential for the anaerobic induction
W box	TTGACC	-382	WRKY转录因子结合位点
			WRKY transcription factor binding site

Cloning of AcWRKY94 gene from kiwifruit and its functional analysis under salt stress

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 9

References 26

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	HU Yingjie, DU Chenqi, WANG Liufan, SHOU Jianxin, WANG Chao, XU Mei, YAN Xu. Research progress of vesicle trafficking in plant response to salt stress [J]. Acta Agriculturae Zhejiangensis, 2025, 37(9): 2003-2011.
[2]	GUAN Xiusheng, LIU Tieshan, WANG Juan, ZHANG Maolin, LIU Chunxiao, DONG Rui, GUAN Haiying, LIU Qiang, XU Yang, HE Chunmei. Bioinformatics analysis and cloning of NF-YA family genes in maize(Zea mays) [J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1605-1614.
[3]	LIU Guomin, ZHENG Xu, LIAO Yujiao, QIN Yexin, QIN Weizhi. Effect of grafting on cold resistance of potato seedlings under low temperature stress [J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1615-1623.
[4]	JIANG Ming, ZHANG Sheng, CHEN Xiaoshang, ZHANG Huijuan. Cloning and functional verification of the gray mold disease responsive gene BoWRKY15 in broccoli(Brassica oleracea var. italica) [J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1723-1732.
[5]	TAN Haixia, PENG Hongli, WANG Lianlong, WEI Jianmei. Differences in soil microbial community diversity between healthy and scab-diseased potato plants in root zone [J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1743-1754.
[6]	TAN Jingru, HU Qizan, YUE Zhichen, TAO Peng, LEI Juanli, LI Biyuan, ZHAO Yanting, ZANG Yunxiang. Comprehensive evaluation system for heat tolerance of seedling-edible Chinese cabbage (Brassica rapa L. ssp.) based on chlorophyll fluorescence parameters [J]. Acta Agriculturae Zhejiangensis, 2025, 37(2): 288-299.
[7]	LIU Xun, XIA Qile, LI Yanpo, WANG Yangguang, LU Shengmin. Optimization of extraction process for soluble and insoluble dietary fibers from Ougan (Citrus suavissima Hort. ex Tanaka) pomace and the differences between their physicochemical properties and functional characteristics [J]. Acta Agriculturae Zhejiangensis, 2025, 37(1): 189-202.
[8]	YU Qinpei, SUN Li, ZHANG Shuwen, YU Zheping, ZHENG Xiliang, QI Xingjiang. Research progress of β-galactosidase in fruits of horticultural crops [J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 2184-2192.
[9]	JIANG Wenjun, SHU Hongsuo, CHEN Zhengman, REN Dianting, YANG Dang, TIAN Rongjiang, DU Zhaokui. Cloning, expression, and bioinformatics analysis of KoWRKY43 gene in Kandelia obovata [J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1832-1843.
[10]	QI Xueli, LI Ying, DUAN Junzhi. Application of salt tolerance genes in wheat salt tolerance genetic engineering [J]. Acta Agriculturae Zhejiangensis, 2024, 36(6): 1447-1457.
[11]	ZHANG Hansheng, CHANG Qinxiang, KANG Jianzhong, LIANG Zongsuo. Research progress on nutritional value and utilization of walnut [J]. Acta Agriculturae Zhejiangensis, 2024, 36(4): 905-919.
[12]	TANG Yuehui, CHEN Shuying, HE Wenqiong, WANG Hanjin, BAO Xinxin, JIA Sainan, WANG Yaoyao, CHEN Yuyang, YANG Tongwen. Cloning and functional analysis of JcERF22 gene from Jatropha curcas [J]. Acta Agriculturae Zhejiangensis, 2024, 36(10): 2219-2228.
[13]	PI Yimeng, LU Yanhui, LYU Zhongxian, XU Yipeng, XU Hongxing. The role of farmland weeds in pest control [J]. Acta Agriculturae Zhejiangensis, 2024, 36(10): 2426-2436.
[14]	QIAO Hongyong, YUAN Tao, ZHAO Xinyong, YANG Huiyan. Characteristics of endophyte’s community changes of Paeonia suffruticosa cv. Lu He Hong fine root in different plant ages [J]. Acta Agriculturae Zhejiangensis, 2024, 36(1): 115-126.
[15]	ZHANG Yi, WANG Feng, CAI Liuti, WANG Hancheng, XIONG Jing, CHEN Xingjiang. Phyllosphere microecology of brown spot and healthy tobacco leaves after application of myclobutanil [J]. Acta Agriculturae Zhejiangensis, 2024, 36(1): 156-167.