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水稻OsPUB4基因克隆、激素诱导表达分析与互作蛋白筛选

李福媛1,**，杨奕2,**，马继琼2，许明辉2，林良斌1,*，孙一丁2,*    

  1. 1 云南农业大学农学与生物技术学院, 云南昆明 650201; 2 云南省农业科学院生物技术与种质资源研究所 / 云南省农业生物技术重点实验室 /农业农村部西南作物基因资源与种质创制重点实验室 / 农业农村部云南稻种资源科学观测实验站, 云南昆明 650205
  • 收稿日期:2024-10-11 修回日期:2025-03-26 接受日期:2025-03-26 网络出版日期:2025-04-01
  • 基金资助:
    本研究由国家自然科学基金项目(32260523)和云南省科技计划项目(202101BD070001?002, 202301BD070001?218, 202405AC350096)资助。

Cloning, hormone-induced expression analysis, and interaction protein screening of OsPUB4 in rice

LI Fu-Yuan1,**,YANG-Yi2,**,MA Ji-Qiong2,XU Ming-Hui2,LIN Liang-Bin1,*,SUN Yi-Ding2,*   

  1. 1 College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China; 2 Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences / Yunnan Provincial Key Laboratory of Agricultural Biotechnology / Southwest Key Laboratory of Crop Gene Resources and Germplasm Creation, Ministry of Agriculture and Rural Affairs / Scientific Observation for Rice Germplasm Resources of Yunnan, Ministry of Agriculture and Rural Affairs, Kunming 650205, Yunnan, China
  • Received:2024-10-11 Revised:2025-03-26 Accepted:2025-03-26 Published online:2025-04-01
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (32260523) and the Science and Technology Program of Yunnan Province (202101BD070001?002, 202301BD070001?218, 202405AC350096). 

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摘要：

为探究E3泛素连接酶OsPUB4的功能，阐明OsPUB4介导的调控机制，以日本晴为材料，克隆水稻U-Box型E3泛素连接酶基因OsPUB4，且通过生物信息学在线网站对启动子顺式作用元件和编码区序列特征进行预测，并构建系统发育树，采用实时荧光定量PCR技术探究该基因在不同植物激素诱导下的表达特征，同时利用酵母cDNA文库对OsPUB4的互作蛋白进行筛选。结果表明：(1) 水稻OsPUB4基因启动子区含多个与激素、光、温度等相关的响应元件，编码区全长2187 bp，无信号肽、含1个跨膜结构域和62个磷酸化位点；(2) OsPUB4与乌拉尔图小麦PUB4蛋白的亲缘关系较近；(3) 通过外源激素诱导发现，短时间内JA会抑制水稻叶片中OsPUB4基因的表达，而IAA则相反；(4) OsPUB4蛋白与OsTPS5、Di19和THIC等存在相互作用关系。综上，OsPUB4基因受外源激素诱导表达，且与多个胁迫响应相关蛋白存在互作关系，为深入研究OsPUB4在水稻胁迫响应方面的功能提供理论依据。

关键词: 水稻, E3泛素连接酶, OsPUB4, 激素诱导表达, 互作蛋白

Abstract:

To investigate the function of the E3 ubiquitin ligase OsPUB4 and elucidate its regulatory mechanism, we cloned the coding sequence of OsPUB4, a U-box-type E3 ubiquitin ligase gene, from the rice cultivar “Nipponbare”. The cis-acting elements of the promoter and the sequence characteristics of the coding region were predicted using bioinformatics tools, and a phylogenetic tree was constructed. The expression patterns of OsPUB4 under different plant hormone treatments were analyzed by quantitative real-time PCR (qRT-PCR). Additionally, interaction proteins of OsPUB4 were identified using a yeast cDNA library screening approach. Our findings revealed that: (1) The promoter region of OsPUB4 contains multiple response elements associated with hormones, light, and temperature. The coding region is 2187 bp in length, lacks a signal peptide, and contains a transmembrane domain and 62 phosphorylation sites. (2) OsPUB4 is closely related to the PUB4 protein of Triticum Urartu. (3) Exogenous jasmonic acid (JA) treatment rapidly suppressed OsPUB4 expression in rice leaves, whereas indole-3-acetic acid (IAA) had the opposite effect. (4) OsPUB4 interacts with OsTPS5, Di19, and THIC. These results suggest that OsPUB4 is induced by exogenous hormones and interacts with multiple stress response-related proteins, providing a theoretical foundation for further investigations into the role of OsPUB4 in rice stress responses.

Key words: rice, E3 ubiquitin ligase, OsPUB4, inducible expression characteristics, interacting proteins

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