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Acta Agronomica Sinica ›› 2025, Vol. 51 ›› Issue (8): 2048-2059.doi: 10.3724/SP.J.1006.2025.54014

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning analysis and functional validation of EfWRKY51 gene related to cold tolerance in Erianthus fulvus

WAN Hui-Lan1,**(), WU Hua-Ying1,4,**(), ZENG Dan1,5, QIAN Zhen-Feng1, ZHAO Chang-Zu1, LIAO Ran-Chao1, HE Li-Lian1,2,*(), LI Fu-Sheng1,2,3,*()   

  1. 1College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    2Sugarcane Research Institute, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    3The Key Laboratory for Crop Production and Smart Agriculture of Yunnan province, Kunming 650201, Yunnan, China
    4Chongqing College of Mobile Communication, Chongqing 400800, China
    5Lijiang City Seed Management Station, Lijiang 674100, Yunnan, China
  • Received:2025-01-26 Accepted:2025-04-27 Online:2025-08-12 Published:2025-05-13
  • Contact: *E-mail: lfs810@sina.com;E-mail: helilan905@sohu.com
  • About author:

    **Contributed equally to this work

  • Supported by:
    Major Science and Technology Project of Yunnan Province(202202AE090021);National Natural Science Foundation of China(31960451)

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Abstract:

WRKY family transcription factors play critical roles in plant response to cold stress. Erianthus fulvus, a wild relative of sugarcane, exhibits remarkable cold tolerance; however, the function of WRKY genes in cold stress its response remains largely unknow. In this study, the EfWRKY51 gene (GenBank accession no: UVJ69259.1) was cloned from E. fulvus. The full-length coding sequence (CDS) was 915 bp and encoding a 304-amino-acid protein. EfWRKY51 expression was upregulated under cold stress, and the encoded protein localized to the nucleus but exhibited no ptranscriptional activation activity. Transgenic tobacco lines overexpressing EfWRKY51 were generated via Agrobacterium-mediated leaf disc transformation. Following cold treatment, transgenic lines showed less leaf wilting compared to the wild type (WT), and displayed significantly higher seed germination rates and root lengths. Moreover, under cold stress, transgenic lines exhibited significantly higher activities of peroxidase (POD) and superoxide dismutase (SOD), as well as increased levels of proline (Pro) and soluble sugars (SS), while malondialdehyde (MDA) content was significantly reduced compared to WT. These results indicate that EfWRKY51 overexpression enhances cold tolerance in transgenic tobacco. This study provides new insights into the regulatory role of EfWRKY51 under cold stress and offers a valuable genetic resource for improving cold tolerance in sugarcane.

Key words: sugarcane, Erianthus fulvus, EfWRKY51 gene, heterologous expression, cold tolerance

Fig. 1

Cloning, bioinformatics and expression analysis of the EfWRKY51 gene A: PCR amplification of EfWRKY51 gene; B: tertiary structure prediction of EfWRKY51 protein; C: multiple sequence alignment of EfWRKY51 protein with other plant WRKY proteins; D: phylogenetic tree of EfWRKY51 protein and other plant WRKY proteins; E: the relative expression of EfWRKY51 gene in different varieties/lines under cold stress. *, ** indicate significant differences at the 0.05 and 0.01 probability levels, respectively; ns: no significant difference."

Fig. 2

Subcellular localization and transcriptional self-activation characteristics of the EfWRKY51 TF A: subcellular localization of EfWRKY51; B: transcriptional self-activation characteristics of EfWRKY51. Scale bar: 10μm."

Fig. 3

Heterologous expression of the EfWRKY51 gene in tobacco A: agrobacterium-mediated transformation of tobacco with EfWRKY51. B: PCR identification of transgenic tobacco lines (B represents the blank control of water amplification, N represents the negative control of WT line amplification, and P represents the positive control of plasmid amplification). C: the relative expression level of EfWRKY51 in transgenic tobacco under cold stress. **, *** indicate significant differences at the 0.01 and 0.001 probability levels, respectively."

Fig. 4

Identification of cold tolerance in tobacco lines overexpressing EfWRKY51 A: germination phenotype of transgenic tobacco seeds after cold stress; B: the germination rate of transgenic tobacco seeds after cold stress; C: root length phenotype of transgenic tobacco after cold stress; D: root length statistics of transgenic tobacco after cold stress; E: cold tolerance phenotype of transgenic tobacco at seedling stage. **, **** indicate significant differences at the 0.01 and 0.0001 probability levels, respectively; ns represents no significant difference."

Fig. 5

Physiological and biochemical responses of tobacco lines overexpressing EfWRKY51 under cold stress *, **, and *** indicate significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively; ns represents no significant difference."

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