Construction of Yeast One-hybrid Library and Screening of Factors Regulating Pvrboh5 Expression in Paspalum vaginatum

doi:10.11733/j.issn.1007-0435.2026.02.007

Acta Agrestia Sinica ›› 2026, Vol. 34 ›› Issue (2): 447-455.DOI: 10.11733/j.issn.1007-0435.2026.02.007

Construction of Yeast One-hybrid Library and Screening of Factors Regulating Pvrboh5 Expression in Paspalum vaginatum

LU Yang, CAI Li-rong, PENG Di, CHEN Ya-fang, LI Jun-xian, LIU Da-lin, PAN Ling

1. Yangzhou University, College of Animal Science and Technology, Yangzhou, Jiangsu Province 225009, China

Received:2025-04-09 Revised:2025-07-13 Published:2026-01-22

海雀稗酵母单杂文库构建及Pvrboh5上游调控因子筛选

陆洋, 蔡丽蓉, 彭迪, 陈亚方, 黎俊显, 刘大林, 潘玲

1. 扬州大学动物科学与技术学院, 江苏扬州 225009

通讯作者: 潘玲，E-mail:panling1199@126.com
作者简介:陆洋（2001-），男，汉族，江苏无锡人，硕士研究生，主要从事海雀稗机理研究，E-mail:lyyzdx2019@163.com；
基金资助:
国家自然科学基金（32401488）；扬州大学海内外优秀人才科研启动经费（137013098/5019）资助

Abstract

Abstract: To explore the molecular regulatory mechanisms of Pvrboh5 expression， this study constructed a yeast one-hybrid library from P. vaginatum （Paspalum vaginatum Swartz） using Gateway technology and screened the upstream regulatory factors of Pvrboh5. Salt-treated stolons of P. vaginatum ‘Sea Isle 2000’ were used as experimental material. The promoter sequence of Pvrboh5 was amplified by PCR and subsequently ligated into the yeast one-hybrid bait vector pBait-AbAi， the construct was named as pPvrboh5-AbAi. This construct was then transformed into the Y1H Gold yeast strain to generate the recombinant bait strain. Concurrently， the yeast one-hybrid library was constructed using Gateway technology. Following mRNA isolation and purification， three reading-frame double-stranded cDNAs were synthesized. Primary libraries were constructed via BP reaction， followed by the generation of high-quality secondary libraries through LR reaction. Finally， transcription factors interacting with Pvrboh5 were identified by screening the P. vaginatum yeast cDNA library. The results showed that transcriptional autoactivation of the pPvrboh5-AbAi/Y1H Gold bait strain was effectively suppressed at an AbA concentration of 100 ng·mL^-1. In the screening experiment， a total of 18 proteins that bind to the Pvrboh5 promoter were identified. These findings provide a theoretical foundation for further elucidating the regulatory mechanisms of the Pvrboh5 gene.

Key words: Paspalum vaginatum, Yeast one-hybrid, Gateway technology, Pvrboh5 promoter, Transcription factor

摘要： 为了探究Pvrboh5表达的分子调控机制，本研究运用gateway技术构建了海雀稗（Paspalum vaginatum Swartz）酵母单杂文库并筛选海雀稗Pvrboh5的上游调控因子。本试验以盐处理‘海岛2000’海雀稗为材料，经PCR扩增得到Pvrboh5的启动子序列，再将其连接至酵母单杂诱饵载体pBait-AbAi上并命名为pPvrboh5-AbAi，接着转化到Y1H Gold酵母菌株上构建重组诱饵菌株。同时利用gateway技术构建酵母单杂文库。在分离纯化mRNA的基础上，合成了3种读码框的双链cDNA，通过BP反应构建其相应的初级文库，再通过LR反应构建高质量的次级文库。最后从海雀稗酵母cDNA文库中筛选与Pvrboh5互作的转录因子。结果显示，pPvrboh5-AbAi/Y1H Gold诱饵菌株在AbA浓度为100 ng·mL^-1时转录自激活得到有效抑制；在筛库实验中，共获得18个与Pvrboh5启动子结合的蛋白。以上结果为后续深入探究Pvrboh5基因调控机制提供理论基础。

关键词: 海雀稗, 酵母单杂, Gateway技术, Pvrboh5启动子, 转录因子

LU Yang, CAI Li-rong, PENG Di, CHEN Ya-fang, LI Jun-xian, LIU Da-lin, PAN Ling. Construction of Yeast One-hybrid Library and Screening of Factors Regulating Pvrboh5 Expression in Paspalum vaginatum[J]. Acta Agrestia Sinica, 2026, 34(2): 447-455.

陆洋, 蔡丽蓉, 彭迪, 陈亚方, 黎俊显, 刘大林, 潘玲. 海雀稗酵母单杂文库构建及Pvrboh5上游调控因子筛选[J]. 草地学报, 2026, 34(2): 447-455.

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Construction of Yeast One-hybrid Library and Screening of Factors Regulating Pvrboh5 Expression in Paspalum vaginatum

海雀稗酵母单杂文库构建及Pvrboh5上游调控因子筛选

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