海雀稗酵母单杂文库构建及Pvrboh5上游调控因子筛选OA

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 subse-quently ligated into the yeast one-hybrid bait vector pBait-AbAi,the construct was named as p Pvrboh5-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 isola-tion 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 p Pvrboh5-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 founda-tion for further elucidating the regulatory mechanisms of the Pvrboh5 gene.