Cloning and Functional Characterization of Cucumber CsWRKY12 Gene in Drought Tolerance

doi:10.13560/j.cnki.biotech.bull.1985.2025-1260

Abstract

Abstract:

Objective WRKY transcription factors play important regulatory roles in plant growth, development, and stress responses. This study aims to investigate the function of the cucumber CsWRKY12 gene in drought stress, providing a basis for elucidating the molecular regulatory mechanisms of cucumber response to drought stress. Method The open reading frame (ORF) of CsWRKY12 was cloned using the cucumber variety ‘9930’ as the material, and its gene and protein sequences were subsequently analyzed. The expression patterns of CsWRKY12 in different cucumber tissues and at different time under drought stress were determined using quantitative real-time PCR (RT-qPCR). Overexpression vectors were constructed and transformed into cucumber hairy roots and Arabidopsis thaliana to explore the function of CsWRKY12 in response to drought stress. Result The CsWRKY12 gene contains an ORF of 939 bp, encoding a 312-amino acid protein with one WRKY domain and a C2H2-type zinc finger domain. Gene structure analysis revealed that CsWRKY12 consists of 4 exons and 3 introns. Phylogenetic analysis showed that CsWRKY12 displays the closest evolutionary relationship to its homologous protein in Benincasa hispida. RT-qPCR results demonstrated that CsWRKY12 is highly expressed in male flowers and leaves, and its expression is significantly upregulated under drought stress. Overexpression of CsWRKY12 in cucumber hairy roots resulted in significantly higher SOD and POD activities under drought stress compared to control, suggesting that CsWRKY12 overexpression may enhance plant drought tolerance. Furthermore, transgenic Arabidopsis plants overexpressing CsWRKY12 were obtained via Agrobacterium-mediated transformation, and the transgenic plants exhibited significantly higher SOD and POD activities, proline content, and expression levels of drought-responsive genes under drought stress, while the relative electrical conductivity and MDA content were significantly lower than those in wild-type (WT) plants. Conclusion CsWRKY12 plays a positive regulatory role in drought stress response by enhancing reactive oxygen species scavenging capacity, promoting the accumulation of osmotic adjustment substances, alleviating membrane lipid peroxidation damage, and regulating the expression of drought-responsive genes.

Key words: cucumber, WRKY gene, CsWRKY12, drought stress, transgenic Arabidopsis, hairy root, functional characterization, expression analysis

LI Chang-yan, XIA Kang-li, WANG Meng, YANG Feng, GUO Xue-ping, HU Zhao-yang, ZHOU Yue-long, LIU Shi-qiang, ZHOU Yong. Cloning and Functional Characterization of Cucumber CsWRKY12 Gene in Drought Tolerance[J]. Biotechnology Bulletin, 2026, 42(11): 124-133.

Figures/Tables 8

Fig. 1 Sequence analyses of the CsWRKY12 gene and its encoded proteinA: Gene structure analysis of CsWRKY12. B: Analysis of the encoded protein sequence of CsWRKY12. The green box represents the WRKY domain, the black line indicates the C2H2 domain, and the conserved C and H residues are marked in red. C: Phylogenetic analysis of CsWRKY12 and its homologous proteins

Fig. 2 Expression patterns of CsWRKY12 gene in different tissues (A) and under different time of drought stress in cucumber (B)Data are means ± SE of three replicates. Different lowercase letters indicate significant differences at P<0.05. The same below

Fig. 3 Analysis of CsWRKY12-overexpressing cucumber hairy roots under drought stressA: Schematic diagram of fluorescence expression in CsWRKY12-GFP hairy root transformation. B: Determination of relative expression levels of CsWRKY12 in cucumber hairy roots. C: Determination of superoxide dismutase (SOD) activity. D: Determination of peroxidase (POD) activity

Fig. 4 Expression levels of the CsWRKY12 gene in overexpression (OE) transgenic Arabidopsis lines

Fig. 5 Seed germination of overexpression (OE) and wild-type (WT) Arabidopsis under drought stressA: Phenotype of seed germination under drought stress simulated by 300 mmol/L mannitol treatment. B: Determination of seed germination rate under drought stress

Fig. 6 Analysis of root length in overexpression (OE) and wild-type (WT) Arabidopsis plants under drought stressA: Root length phenotype of Arabidopsis plants under drought stress simulated by 200 mmol/L mannitol treatment. B: Determination of the relative root length of Arabidopsis plants under drought stress

Fig. 7 Phenotype (A) and stress-related physiological indices (B-G) in overexpression (OE) and wild-type (WT) Arabidopsis plants under drought stress

Fig. 8 Expression of stress related-genes in overexpression (OE) and wild-type (WT) Arabidopsis plants under drought stress

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