花生转录因子AhZHD9的生物信息学分析及耐盐性研究

doi:10.13560/j.cnki.biotech.bull.1985.2026-0315

摘要/Abstract

摘要：

目的鉴定并解析花生ZHD转录因子AhZHD9的结构特征及其在盐胁迫响应中的功能，探讨其在植物抗盐调控中的潜在作用，为解析花生ZHD转录因子参与逆境响应的分子机制及作物抗盐分子育种提供理论依据。方法以花生AhZHD9基因为研究对象，通过生物信息学方法分析其蛋白结构特征、互作蛋白及启动子顺式作用元件，并进行亚细胞定位分析；利用RT-qPCR检测其在盐胁迫下的表达模式；构建AhZHD9过表达拟南芥，通过测定叶绿素和MDA含量及SOD、CAT和POD活性，以及植株表型评价其抗盐功能。结果 AhZHD9蛋白含有典型的锌指结构域（ZF）和同源异型盒结构域（HD），属于ZHD转录因子家族成员。蛋白结构预测结果表明，AhZHD9呈现“结构域有序、调控区无序”的结构特征。蛋白互作预测结果显示，AhZHD9可能与含有bHLH、zf-CW、MBD、REF及Senescence_reg等结构域的蛋白发生互作。启动子分析表明，AhZHD9启动子中含有多种参与逆境胁迫响应和激素信号调控的顺式作用元件；亚细胞定位结果显示，该蛋白主要定位于细胞核。表达分析进一步发现，AhZHD9在盐胁迫条件下被显著诱导表达。功能验证结果表明，与野生型相比，AhZHD9过表达拟南芥在盐胁迫下具有更低的MDA含量和更高的叶绿素含量，同时SOD、CAT和POD活性也显著增强，且AhZHD9过表达拟南芥株系生长状态优于野生型，说明过表达AhZHD9能够增强植株抗氧化能力并减轻盐胁迫诱导的氧化损伤。结论 AhZHD9是一个参与盐胁迫响应的ZHD转录因子，其过表达能够增强植物抗氧化防御能力并提高盐耐受性。

关键词: 花生, ZHD转录因子, 生物信息学, 盐胁迫, 抗氧化酶, 相对表达量, 亚细胞定位

Abstract:

Objective To identify and characterize the structural features of the peanut ZHD transcription factor AhZHD9 and elucidate its function in salt stress response, thereby exploring its potential role in regulating plant salt tolerance and providing a theoretical basis for understanding the molecular mechanisms of peanut ZHD transcription factors in stress response and for molecular breeding of salt-tolerant crops. Method Using peanut AhZHD9 as the target gene, bioinformatics approaches were employed to analyze its protein structural features, interacting proteins, and promoter cis-acting elements, followed by subcellular localization analysis. The expression pattern of AhZHD9 under salt stress was detected by RT-qPCR. AhZHD9-overexpressing Arabidopsis lines were constructed, and salt tolerance was evaluated by measuring chlorophyll and MDA contents, SOD, CAT, and POD activities, as well as plant phenotypes. Result The AhZHD9 protein contains typical zinc finger (ZF) and homeodomain (HD) domains, classifying it as a member of the ZHD transcription factor family. Protein structure prediction revealed that AhZHD9 exhibits a structural pattern of “ordered domains and disordered regulatory regions”. Protein-protein interaction prediction indicated that AhZHD9 may interact with proteins containing bHLH, zf-CW, MBD, REF, and Senescence_reg domains. Promoter cis-acting element analysis revealed that the AhZHD9 promoter region contains multiple elements related to abiotic stress and hormone signaling. Subcellular localization analysis showed that AhZHD9 protein is predominantly localized in the nucleus. Expression analysis demonstrated that AhZHD9 expression was significantly up-regulated under salt stress treatment. Functional validation showed that under salt stress conditions, AhZHD9-overexpressing Arabidopsis lines exhibited significantly lower MDA content but higher chlorophyll content, as well as significantly higher SOD, CAT, and POD activities compared to wild-type plants. Moreover, AhZHD9-overexpressing lines displayed less severe growth inhibition than wild-type plants, indicating that AhZHD9 overexpression can enhance antioxidant capacity and mitigate oxidative damage induced by salt stress. Conclusion AhZHD9 is a ZHD transcription factor involved in salt stress response, and its overexpression can enhance plant antioxidant defense capacity and improve salt tolerance.

Key words: peanut, ZHD transcription factor, bioinformatics, salt stress, antioxidant enzymes, relative expression level, subcellular localization

任文斌, 陈登科, 吴翠翠. 花生转录因子AhZHD9的生物信息学分析及耐盐性研究[J]. 生物技术通报, 2026, 42(9): 297-305.

REN Wen-bin, CHEN Deng-ke, WU Cui-cui. Bioinformatics Analysis and Salt Tolerance Study of the Peanut Transcription Factor AhZHD9[J]. Biotechnology Bulletin, 2026, 42(9): 297-305.

图/表 7

图1 AhZHD9编码区（CDS）克隆及结构分析A：花生青花6号叶片中扩增1 023 bp的AhZHD9编码序列；B：AhZHD9基因结构分析；C：AhZHD9蛋白保守结构域预测

Fig. 1 Cloning and structural analysis of the AhZHD9 CDSA: Amplification of the 1 023 bp coding sequence of AhZHD9 from leaves of peanut cultivar Qinghua 6. B: Gene structure analysis of AhZHD9. C: Conserved domain prediction of the AhZHD9 protein

图2 AhZHD9蛋白互作网络分析

Fig. 2 Protein-protein interaction network analysis of AhZHD9

图3 AhZHD9启动子区顺式作用元件分析A：AhZHD9基因顺式作用元件分布；B：AhZHD9基因顺式作用元件类型与数量统计

Fig. 3 Analysis of cis-acting elements in the AhZHD9 promoter regionA: Distribution of cis-acting elements in AhZHD9 gene. B: Statistics of cis-acting element types and quantities in the AhZHD9 gene

图4 AhZHD9的亚细胞定位Bright field为普通光学显微镜下获得的细胞整体结构图像；YFP表示黄色荧光信号；Fluorescence表示红色荧光信号；Merged为前三幅图像荧光信号叠加后的合并图像。比例尺：50 μm

Fig. 4 Subcellular localization of AhZHD9Bright field: Overall cellular structure observed under a light microscope. YFP: Yellow fluorescent signal. Fluorescence: Red fluorescent signal. Merged: Merged image of the three fluorescence signals above. Scale bar: 50 μm

图5 AhZHD9过表达株系的盐胁迫根长实验*，**分别表示在0.05、0.01水平上差异显著；WT表示野生型拟南芥，OE表示AhZHD9过表达株系。下同

Fig. 5 Root length assay of AhZHD9-overexpressing lines under salt stress* and ** indicate significant differences at the levels of 0.05 and 0.01, respectively. WT indicates wild-type Arabidopsis plants, and OE indicates AhZHD9-overexpressing lines. The same below

图6 AhZHD9过表达株系在盐胁迫下的相对表达量

Fig. 6 Relative expression analysis of AhZHD9-overexpressing lines under salt stress

图7 AhZHD9过表达拟南芥在盐胁迫下的表型及生理指标分析A：盐胁迫处理前及处理12 d后的植株表型；B：叶绿素含量；C：丙二醛（MDA）含量；D：过氧化物酶（POD）活性；E：超氧化物歧化酶（SOD）活性；F：过氧化氢酶（CAT）活性

Fig. 7 Phenotypic and physiological analysis of AhZHD9-overexpressing Arabidopsis under salt stressA: Plant phenotype before salt treatment and after 12 d of salt stress. B: Chlorophyll content. C: Malondialdehyde (MDA) content. D: Peroxidase (POD) activity. E: Superoxide dismutase (SOD) activity. F: Catalase (CAT) activity

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