Cloning of the gene ZjWRKY63 from Zoysia japonica and its salt resistance analysis in transgenic Arabidopsis

doi:10.11686/cyxb2025014

Abstract

Abstract:

WRKY transcription factors play crucial regulatory roles in plant growth and development， pathogen defense， and responses to abiotic stress. Based on the previous gene family identification of WRKY transcription factor genes in Zoysia japonica， as well as the transcriptome analysis of the salt-tolerant and salt-sensitive Z. japonica species under salt stress， a salt stress-responsive WRKY transcription factor gene named ZjWRKY63 was screened out. To further investigate the salt-tolerance function of this gene， primers were designed for PCR amplification， and the coding sequence （CDS） with an open reading frame of 921 bp was cloned. The ZjWRKY63 encodes a protein containing 306 amino acids， which possesses one WRKY functional conserved domain and one C₂H₂ zinc finger structural motif， and belongs to theWRKY IIa subfamily. Bioinformatics analysis revealed that the maximum score value of the average signal peptide for the amino acid residues of ZjWRKY63 was 0.267， indicating that this protein does not possess a signal peptide. Hydrophobicity prediction analysis showed that the grand average of hydrophobicity （GRAVY） value was -0.395， indicating that this protein is a hydrophilic protein. Transmembrane region prediction found no transmembrane region in ZjWRKY63. Prediction of subcellular localization showed that ZjWRKY63 would be localized in the nucleus. Phylogenetic analysis demonstrated that ZjWRKY63 of Z. japonica has the closest genetic relationship with the Persea americana， PaWRKY65. Salt stress treatment was conducted using the seeds and plants of transgenic Arabidopsis thaliana with overexpressed ZjWRKY63. It was found that the seed germination rate， survival rate and number of lateral roots of the overexpressing plants were significantly higher than those of the wild type. Meanwhile， in the transgenic A. thaliana with ZjWRKY63， the expression of genes related to salt stress response was significantly higher than that in the wild type in all cases. We speculate that the ZjWRKY63 gene enhances the salt tolerance of transgenic A. thaliana by regulating the expression of stress response genes. The above research results provide preliminary confirmation of the salt-tolerance function of the ZjWRKY63 gene， and lay a scientific foundation for further investigation of the molecular mechanism of the salt-tolerance conferred by the ZjWRKY63 gene.

Key words: Zoysia japonica, ZjWRKY63 gene, gene cloning, salt tolerance, molecular mechanism

Yong-long LI, Sheng-hui ZHOU, Meng-yao XUE, Yuan GAO, Le JU, Yi-bing CHEN, Song-lin FU, Jian-hao HAO, Heng LI, Kun ZHANG, Zhi-fang ZUO. Cloning of the gene ZjWRKY63 from Zoysia japonica and its salt resistance analysis in transgenic Arabidopsis[J]. Acta Prataculturae Sinica, 2025, 34(12): 157-169.

Figures/Tables 8

References 38

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名称Name	寡核苷酸Oligonucleotides （5′-3′）	用途Purpose
ZjWRKY63-F	GTGTTACTTCCCCGGGGATCCATGGACAGCGAGTGGAGCG	ZjWRKY63基因克隆 For the gene cloning of ZjWRKY63
ZjWRKY63-R	GTCATCCTTGTAGTCAAGCTTTGTCATGGCAGTGCCTCC	ZjWRKY63基因克隆 For the gene cloning of ZjWRKY63
AtABI5-F	AGAGGGATAGCGAACGAGTCTAGTC	用于下游基因的qRT-PCR表达分析 For the qRT-PCR of downstream gene expression analysis
AtABI5-R	GTTCGGGTTTGGATTAGGTTTAGG
AtCOR15A-F	CAGTTCGTCGTCGTTTCT
AtCOR15A-R	CCAATGTATCTGCGGTTT
AtDREB1A-F	AGGAGACGTTGGTGGAGGCT
AtDREB1A-R	ACGTCGTCATCATCGCCGTC
AtMYC2-F	GCGTTGATGGATTTGGAGTT
AtMYC2-R	TTGCTCTGAGCTGTTCTTGC
AtSOS1-F	TCGTTTCAGCCAAATCAGAAAGT
AtSOS1-R	TTTGCCTTGTGCTGCTTTCC
AtSOS2-F	GGCTTGAAGAAAGTGAGTCTCG
AtSOS2-R	GCTACATAGTTCGGAGTTCCACA
AtACT-F	GGTAACATTGTGCTCAGTGGTGG	拟南芥内参基因 Inner reference gene of A. thaliana
AtACT-R	AACGACCTTAATCTTCATGCTGC	拟南芥内参基因 Inner reference gene of A. thaliana

名称Name	寡核苷酸Oligonucleotides （5′-3′）	用途Purpose
ZjWRKY63-F	GTGTTACTTCCCCGGGGATCCATGGACAGCGAGTGGAGCG	ZjWRKY63基因克隆 For the gene cloning of ZjWRKY63
ZjWRKY63-R	GTCATCCTTGTAGTCAAGCTTTGTCATGGCAGTGCCTCC	ZjWRKY63基因克隆 For the gene cloning of ZjWRKY63
AtABI5-F	AGAGGGATAGCGAACGAGTCTAGTC	用于下游基因的qRT-PCR表达分析 For the qRT-PCR of downstream gene expression analysis
AtABI5-R	GTTCGGGTTTGGATTAGGTTTAGG
AtCOR15A-F	CAGTTCGTCGTCGTTTCT
AtCOR15A-R	CCAATGTATCTGCGGTTT
AtDREB1A-F	AGGAGACGTTGGTGGAGGCT
AtDREB1A-R	ACGTCGTCATCATCGCCGTC
AtMYC2-F	GCGTTGATGGATTTGGAGTT
AtMYC2-R	TTGCTCTGAGCTGTTCTTGC
AtSOS1-F	TCGTTTCAGCCAAATCAGAAAGT
AtSOS1-R	TTTGCCTTGTGCTGCTTTCC
AtSOS2-F	GGCTTGAAGAAAGTGAGTCTCG
AtSOS2-R	GCTACATAGTTCGGAGTTCCACA
AtACT-F	GGTAACATTGTGCTCAGTGGTGG	拟南芥内参基因 Inner reference gene of A. thaliana
AtACT-R	AACGACCTTAATCTTCATGCTGC	拟南芥内参基因 Inner reference gene of A. thaliana