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作物学报 ›› 2025, Vol. 51 ›› Issue (8): 2048-2059.doi: 10.3724/SP.J.1006.2025.54014

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

蔗茅耐寒相关基因EfWRKY51克隆分析及功能验证

万慧兰1,**(), 吴华英1,4,**(), 曾丹1,5, 钱禛锋1, 赵昌祖1, 廖然超1, 何丽莲1,2,*(), 李富生1,2,3,*()   

  1. 1云南农业大学农学与生物技术学院, 云南昆明 650201
    2云南农业大学甘蔗研究所, 云南昆明 650201
    3云南省作物生产与智慧农业重点实验室, 云南昆明 650201
    4重庆移通学院, 重庆 400800
    5丽江市种子管理站, 云南丽江 674100
  • 收稿日期:2025-01-26 接受日期:2025-04-27 出版日期:2025-08-12 网络出版日期:2025-05-13
  • 通讯作者: *李富生, E-mail: lfs810@sina.com;何丽莲, E-mail: helilan905@sohu.com
  • 作者简介:万慧兰, E-mail: whl118139@sina.com;
    吴华英, E-mail: 1997wuhuaying@sina.com

    **同等贡献

  • 基金资助:
    云南省重大科技专项(202202AE090021);国家自然科学基金项目(31960451)

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 Published:2025-08-12 Published online: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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摘要：

WRKY家族基因广泛参与植物的低温胁迫应答, 是植物中最重要的转录因子之一。蔗茅属于甘蔗的近缘野生种, 具有优异的耐寒性, 但WRKY基因是否参与蔗茅对低温胁迫的应答尚不清楚。本研究从蔗茅中克隆得到EfWRKY51基因(GenBank: UVJ69259.1), 该基因CDS全长915 bp, 编码304个氨基酸。EfWRKY51基因被冷胁迫诱导上调表达, 编码蛋白亚细胞定位于细胞核中, 且无转录自激活活性。通过农杆菌介导的叶盘法转化, 获得异源表达EfWRKY51基因的转基因烟草株系。低温胁迫处理后, 转基因烟草株系的叶片萎蔫程度低于野生型, 且种子发芽率和根长均显著高于野生型(WT); 此外, 低温胁迫下转基因株系的抗氧化酶过氧化物酶(POD)、超氧化物歧化酶(SOD)活性以及渗透调节物质脯氨酸(Pro)、可溶性糖(SS)含量均显著高于WT株系, 丙二醛(MDA)含量显著低于WT株系, 表明EfWRKY51基因过表达增强了转基因烟草株系的耐寒性。本研究结果为阐明EfWRKY51基因调控蔗茅耐寒性提供了依据, 为甘蔗耐寒遗传改良提供了基因资源。

关键词: 甘蔗, 蔗茅, EfWRKY51基因, 异源表达, 耐寒性

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

图1

EfWRKY51基因的克隆及生物信息学和表达量分析 A: EfWRKY51基因的PCR扩增; B: EfWRKY51基因编码蛋白的三级结构预测; C: EfWRKY51蛋白与其他植物WRKY蛋白的多序列比对; D: EfWRKY51蛋白与其他植物WRKY蛋白的系统发育树; E: EfWRKY51基因在不同品种/系中对冷胁迫的相对表达量。*、**分别表示在0.05、0.01水平差异显著; ns表示差异不显著。"

图2

EfWRKY51转录因子的亚细胞定位和转录自激活特性 A: EfWRKY51转录因子的亚细胞定位; B: EfWRKY51转录因子的转录自激活特性。标尺为10μm。"

图3

EfWRKY51基因在烟草中异源表达 A: 农杆菌介导EfWRKY51的烟草转化。B: 转基因烟草株系的PCR扩增鉴定; B代表空白对照, N代表阴性对照, P代表阳性对照。C: EfWRKY51在转基因烟草中的相对表达量。**、***分别表示在0.01、0.001水平差异显著。"

图4

过表达EfWRKY51烟草株系的耐寒性鉴定 A: 冷胁迫后转基因烟草种子的发芽情况; B: 冷胁迫后转基因烟草种子的发芽率; C: 冷胁迫后转基因烟草的根长表型; D: 冷胁迫后转基因烟草的根长统计; E: 转基因烟草苗期耐冷性表型。**、****分别表示在0.01、0.0001水平差异显著; ns代表无显著差异。"

图5

低温胁迫下过表达EfWRKY51烟草株系的生理生化参数表现 *、**和***分别表示在0.05、0.01和0.001水平差异显著; ns代表无显著差异。"

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