Bioinformatics Analysis and Salt Tolerance Study of the Peanut Transcription Factor AhZHD9

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

Abstract

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

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.

Figures/Tables 7

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

Fig. 2 Protein-protein interaction network analysis of AhZHD9

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

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

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

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

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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