Acta Agrestia Sinica

Acta Agrestia Sinica ›› 2024, Vol. 32 ›› Issue (1): 66-74.DOI: 10.11733/j.issn.1007-0435.2024.01.007

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Cloning of SgPAL3 Gene Promoter and Screening of Upstream Transcription Factors in Stylosanthes

DAI Rong-hui1,2, GAO Meng-ze1,2, WANG Fang1,2, JIANG Ling-yan1,2, LUO Li-juan1,2   

  1. 1. School of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan Province 570228, China;
    2. Sanya Institute of Breeding and Multipication, Hainan University, Sanya, Hainan Province 572025, China
  • Received:2023-08-14 Revised:2023-10-02 Online:2024-01-15 Published:2024-01-30

柱花草SgPAL3基因启动子的克隆及上游转录因子的筛选

戴镕徽1,2, 高梦泽1,2, 王芳1,2, 蒋凌雁1,2, 罗丽娟1,2   

  1. 1. 海南大学热带农林学院, 海南 海口 570228;
    2. 海南大学三亚南繁研究院, 海南 三亚 572025
  • 通讯作者: 蒋凌雁,E-mail:lyjiang@hainanu.edu.cn
  • 作者简介:戴镕徽(1999-),女,汉族,黑龙江富锦人,硕士研究生,主要从事抗病相关基因克隆与功能研究,E-mail:dairh1999@163.com
  • 基金资助:
    国家自然科学基金项目（32201449，31872409）；海南大学协同创新中心项目（XTCX2022NYC02）；海南省自然科学基金高层次人才项目（320RC466）；海南省科技专项（ZDYF2020211）资助

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Abstract: Stylosanthes (stylo) is an important leguminous forage widely grown in tropical areas. Anthracnose is a serious disease affecting the growth of stylo. Previous studies show that SgPAL3 gene of stylo has function in the resistance against Colletotrichum gloeosporipides. In this study, the promoter regions of -2 000 to 0 bp and -1 500 to 0 bp were cloned into bait vectors, and the concentration of AbA that can inhibit the self-activation of bait vectors was determined. The results showed that 500 ng·mL-1 (Aureobasidin A, AbA) inhibited the self-activation of both bait vectors. The yeast cDNA library of stylo in response to C. gloeosporioides infection was constructed using the Gateway technology. The capacity of the library was 1.20×107. The inserted fragments were mainly distributed from 750 to 2 000 bp, and the recombination rate was 100%. The transcription factors interacting with SgPAL3 gene promoter were screened by yeast one-hybrid technique, and the the interactions of three transcription factors (SgASIL2, SgHAT5 and SgZHD8) with the SgPAL3 promoter were verified by yeast point-to-point experiments. The qRT-PCR analysis showed that the expression of SgASIL2, SgHAT5 and SgZHD8 were all responsive to C. gloeosporioides infection, indicating the these three transcription factors involved in regulating the defense responses of stylo against C. gloeosporioides. These results would lay a foundation for further research on transcriptional regulation mechanisms of SgPAL3 in response to C. gloeosporioides infection.

Key words: Stylosanthes, Promoter, Yeast one-hybrid, Transcription factors, SgPAL3 gene

摘要： 柱花草（Stylosanthes spp.）是热带地区广泛种植的重要草种，炭疽病是危害柱花草的严重病害，柱花草SgPAL3基因具有抗炭疽菌的功能。本研究对启动子区域-2 000~0 bp，-1 500~0 bp序列分别构建诱饵载体并检测自激活，结果表明500 ng·mL-1金担子素（Aureobasidin A，AbA）可以抑制两种诱饵载体的自激活。利用Gateway技术构建了柱花草响应炭疽菌的酵母cDNA文库，其容量为1.20×107，插入片段主要分布在750~2 000 bp，重组率为100%；利用酵母单杂交技术，筛选与SgPAL3基因启动子互作的转录因子，并验证了3个转录因子（SgASIL2，SgHAT5和SgZHD8）与SgPAL3启动子的互作关系；qRT-PCR分析表明，SgASIL2，SgHAT5和SgZHD8均响应炭疽菌的侵染，说明它们可能调控柱花草对炭疽病的抗性。本研究为进一步解析SgPAL3响应炭疽菌侵染的转录调控机制奠定了基础。

关键词: 柱花草, 启动子, 酵母单杂, 转录因子, SgPAL3基因

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