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.

Paspalum vaginatum  /  Yeast one-hybrid  /  Gateway technology  /  Pvrboh5 promoter  /  Transcription factor