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

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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

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