黄瓜CsWRKY12基因的克隆及抗旱功能鉴定

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

摘要/Abstract

摘要：

目的 WRKY转录因子在植物生长发育及抵御胁迫过程中具有重要调控作用。探究黄瓜CsWRKY12基因在干旱胁迫中的功能，为解析黄瓜响应干旱胁迫的分子调控机制提供基础。方法以黄瓜品种‘9930’为材料，克隆CsWRKY12基因的开放阅读框，并对其基因和蛋白序列进行分析。通过实时荧光定量PCR技术分析CsWRKY12在黄瓜不同组织及干旱胁迫不同时间的表达模式。构建过表达载体，分别转化黄瓜毛状根和拟南芥，探索CsWRKY12基因在干旱胁迫中的功能。结果 CsWRKY12基因的开放阅读框为939 bp，编码312个氨基酸的蛋白质，其含有1个WRKY结构域和C2H2型锌指结构。基因结构显示该基因由4个外显子和3个内含子组成。系统进化分析表明，CsWRKY12和冬瓜中的同源蛋白亲缘关系最近。RT-qPCR结果显示，CsWRKY12在黄瓜的雄花和叶片中表达量较高，且干旱处理可显著诱导其表达上调。在黄瓜毛状根中过表达CsWRKY12发现，过表达毛状根的SOD和POD活性在干旱胁迫后明显高于对照，暗示过表达CsWRKY12可提高植物抗旱性。进一步通过农杆菌介导的方法获得过表达CsWRKY12的拟南芥转基因植株，发现转基因植株在干旱处理下，SOD和POD活性明显提升，脯氨酸含量和干旱响应基因的表达明显高于野生型，而相对电导率和MDA含量则明显低于野生型植株。结论 CsWRKY12可通过增强活性氧清除能力、促进渗透调节物质积累、减轻膜脂过氧化损伤以及调控干旱响应基因的表达，在黄瓜抵御干旱胁迫过程中发挥正向调控作用。

关键词: 黄瓜, WRKY基因, CsWRKY12, 干旱胁迫, 转基因拟南芥, 毛状根, 功能鉴定, 表达分析

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

李昌焱, 夏康力, 王蒙, 杨凤, 郭学平, 胡朝阳, 周跃龙, 刘世强, 周勇. 黄瓜CsWRKY12基因的克隆及抗旱功能鉴定[J]. 生物技术通报, 2026, 42(11): 124-133.

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.

图/表 8

图1 CsWRKY12基因及其编码蛋白序列分析A：CsWRKY12基因结构分析；B：CsWRKY12编码蛋白序列分析；绿色方框为WRKY结构域，横线为C2H2结构域，保守的C和H用红色标记；C：CsWRKY12及其同源蛋白的系统进化分析

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

图2 CsWRKY12在黄瓜不同组织（A）及在干旱胁迫不同时间（B）的表达模式数据为3个重复的平均值±SE。不同小写字母代表显著性水平（P<0.05）。下同

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

图3 过表达CsWRKY12黄瓜毛状根干旱胁迫分析A：CsWRKY12-GFP毛状根转化荧光表达示意图；B：毛状根CsWRKY12相对表达量测定；C：SOD活性的测定；D：POD活性的测定

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

图4 转基因拟南芥株系中CsWRKY12基因的表达水平

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

图5 CsWRKY12过表达（OE）和野生型（WT）拟南芥在干旱胁迫下的萌发情况A：300 mmol/L甘露醇处理模拟干旱胁迫下种子萌发表型；B：干旱胁迫下的种子萌发率测定

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

图6 CsWRKY12过表达（OE）和野生型（WT）拟南芥植株在干旱胁迫下的根长伸长情况A：200 mmol/L甘露醇处理模拟干旱胁迫下拟南芥植株根长表型；B：干旱胁迫下拟南芥植株相对根长的测定

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

图7 CsWRKY12过表达（OE）和野生型（WT）拟南芥植株在干旱胁迫下的表型（A）和逆境生理指标测定（B-G）

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

图8 干旱胁迫下CsWRKY12过表达（OE）和野生型（WT）拟南芥植株胁迫响应相关基因表达量

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

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