Abstract: Previously， the specifically differentially expressed gene ZmMKK3 responding to high‑temperature stress in heat‑tolerant maize cultivar Zhengdan 819 was identified from transcriptome data of Zhengdan 819 and its parental lines before and after high temperature stress. Building on this，bioinformatics methods were employed to predict the structural domains，conserved motifs，and secondary structure of ZmMKK3 protein. Maize protoplasts were extracted to determine the subcellular localization of ZmMKK3 protein. The effects of 100 μmol/L abscisic acid（ABA）on heat tolerance，proline content，antioxidant enzyme activity of maize seedlings，and the expression patterns of ZmMKK3 and ZmSNRK2.3 genes were analyzed under high temperature（42 ℃）. Interacting proteins of ZmMKK3 were predicted，and the molecular mechanism of ZmMKK3 in response to high temperature stress was preliminarily explored，so as to provide genetic resources for breeding heat‑tolerant maize varieties.The results showed that ZmMKK3 didn’t have transmembrane domain.The secondary structure of ZmMKK3 protein primarily consisted of α‑helices（36.01%），β‑sheets（10.69%），and disordered regions（53.30%）.ZmMKK3 was primarily localized in the nucleus and cytoplasm. Under high temperature，ABA application enhanced chlorophyll content，relative water content，proline content，and antioxidant enzymeactivity［superoxide dismutase（SOD），peroxidase（POD），catalase（CAT）］in maize seedling leaves，thereby improving plant heat tolerance.ZmMKK3 gene was induced by both high temperature and ABA，its expression was stronger with ABA application under high temperature，and the expression pattern of ZmSnRK2.3 gene in the ABA signaling pathway aligned with that of ZmMKK3，indicating that ZmMKK3 induced by ABA participated in the response to high temperature stress.ZmMKK3 interacted with PP2C9（protein phosphatase 9）in the ABA signaling pathway and B4FZ26（a WD40‑repeat domain protein）associated with miRNA biogenesis，and the interaction coefficients were 0.534 and 0.518，respectively.Collectively，these findings suggest that ZmMKK3 likely responds to maize high temperature stress through the ABA‑MPK signaling pathway.

Key words: Maize, MKK3, High temperature stress, Molecular mechanism, ABA?MPK

摘要： 前期利用耐热性较强的玉米栽培种郑单819及其父母本高温前后的转录组数据鉴定到郑单819中特异的响应高温胁迫的差异表达基因ZmMKK3。在此基础上，利用生物信息学方法预测ZmMKK3蛋白的结构域、保守基序和二级结构，提取玉米原生质体进行ZmMKK3蛋白的亚细胞定位，分析高温（42 ℃）下加入100 μmol/L 脱落酸（ABA）对玉米幼苗耐热性、脯氨酸含量、抗氧化酶活性的影响及ZmMKK3和ZmSNRK2.3基因的表达情况，并预测ZmMKK3的互作蛋白，初步探讨ZmMKK3响应高温胁迫的分子机制，为培育耐热玉米品种提供基因资源。结果表明，ZmMKK3不具有跨膜结构域。ZmMKK3蛋白二级结构主要为α螺旋（36.01%）、β折叠（10.69%）和无规则卷曲（53.30%）。ZmMKK3主要定位在细胞核和细胞质中。高温下加入ABA可以提高玉米幼苗叶片叶绿素含量、相对含水量、脯氨酸含量和抗氧化酶［超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）］活性，从而提高植株的耐高温能力。ZmMKK3基因受高温胁迫和ABA诱导表达，并且高温胁迫下加入ABA诱使ZmMKK3基因表达更强烈，ABA 信号通路中的ZmSnRK2.3基因的表达模式与ZmMKK3基因一致，表明ZmMKK3基因受ABA诱导表达参与植物对高温胁迫的响应过程。ZmMKK3与ABA 信号途径中的蛋白磷酸酶9（PP2C9）、miRNA发生相关的WD40重复结构域蛋白B4FZ26互作，互作系数分别为0.534、0.518。综上，初步推测ZmMKK3通过ABA-MPK信号通路响应玉米的高温胁迫。

关键词: 玉米, MKK3, 高温胁迫, 分子机制, ABA-MPK