The Research On Effects Of ERF2 Transcription Factor Regulates Expression Of CCD1 And CCD4 Genes In Osmanthus Fragrans

Osmanthus fragrans Lour,is well known as Osmanthus,which is belonged to Oleaceae and native to China.It is a special evergreen broad-leaved shrub or small tree with economic value,and it has a cultivation history more than 2500 years.O.fragrans has been emphasised by people due to has a wide range of applications in ornamental,food,medicinal and spices.According to the characteristics of flowering period,flower color and fragrance,O.fragrans can be usually divided into five groups including Luteus,Asiaticus,Albus,Auranticus and Colour group.The previous studies indicated that the color of the petals of O.fragrans is positive correlation with the carotenoid content.Carotenoid cleavage dioxygenase(CCD)CCD1 and CCD4 can dissociate carotenoids and produce the aromatic substance ionone(including α-ionone and β-ionone),which makes it have a unique flavor.Therefore,CCD1 and CCD4 are the key genes in the cleavage of O.fragrans carotenoids and the production of ionone.In present study,we uses molecular biology methods to study the regulation of O.fragrans transcription factor ERF2 on the expression of genes CCD1 and CCD4 in O.fragrans.The main results are as follows:(1)Cloning of gene ERF2 from O.fragrans and analysis of expression patterns of genes CCD1,CCD4 and ERF2The specific primer was designed to amplify the gene ERF2,which has a full-length coding region of 732 bp and encodes 244 amino acids.The Blast alignment showed that ERF2 contains an AP2 domain with high homology to the ERF2 amino acid sequence of Olea europaea,Sesamum indicum,and Nicotiana tabacum.O.fragrans ERF2 protein has a closer relationship with Olea europaea.The results of RT-PCR showed that the expression patterns of CCD1,CCD4 and ERF2 were identical in the petals of Luteus and Auranticus in different flowering stages.It is speculated that transcription factor ERF2 may be involved in the regulation of genes CCD1 and CCD4 expression.(2)Subcellular localization of transcription factor ERF2The 35S::ERF2 recombinant expression vector was constructed and transformed into tobacco by Agrobacterium-mediated transient transformation technique,using the 35S::GFP vector as a control.After incubation at 25°C for 3 days,it was observed under a fluorescence microscope.It was found that green fluorescence in the control leaf cells was mainly concentrated in the cytoplasm,while green fluorescence in the ERF2 overexpressing leaf cells was concentrated in the nucleus,indicating that the transcription factor ERF2 is localized in the nucleus.(3)Overexpression of gene ERF2 in tobacco leaves promoted the expression of genes CCD1 and CCD4 in tobaccoThe 35S::ERF2 and 35S::GFP vectors were transformed into tobacco by Agrobacterium transient transformation method.After incubation at 25°C for 3 days,RNA in leaves was extracted,and the expression levels of genes CCD1 and CCD4 were detected by fluorescence quantitative PCR.The results shows that overexpression of gene ERF2 can promotes the expression of genes CCD1 and CCD4.(4)Interaction analysis of transcription factor ERF2 and related components of genes CCD1 and CCD4 promotersThe 35S::ERF2 overexpression vector and the CCD1pro::GUS and CCD4pro::GUS recombinant vectors were transformed into tobacco by Agrobacterium transient transformation,35S::GFP+CCD1pro::GUS and 35S::GFP+CCD4pro::GUS vector combination was used as a control.After cultured at 25°C for 3 days,the GUS protein activity and gene GUS expression in tobacco leaves were detected by histochemical staining and real-time PCR.The results showed that the control leaves showed weak blue color,while the ERF2 overexpressed tobacco leaves showed the expression of GUS gene in tobacco leaves,showing darker blue color,and ERF2 overexpression was significantly higher than that of the control group.The study indicates that the transcription factor ERF2 can bind to the genes CCD1 and CCD4 promoter-related elements to up-regulating gene GUS expression.(5)Prokaryotic expression and purification of OfERF2 proteinThe prokaryotic expression vector pET-30a-ERF2 was constructed and transfected into BL21 competent cells.After induction by IPTG,the 35 KD-sized ERF2 protein band was detected by SDS-polyacrylamide gel electrophoresis,and ERF2 protein was purified.(6)EMSA validates the interaction of ERF2 protein with related elements on the genes CCD1 and CCD4 promotersThe transcription factor ERF2 binding element containing the genes CCD1 and CCD4 promoters was synthesized in vitro,and the interaction between ERF2 protein and related elements was verified by EMSA experiment.The results showed that a distinct retention band appeared when the ERF2 protein and the fluorescently labeled element were simultaneously electrophoresed,indicating that the ERF2 protein can bind to the transcription factor ERF binding element on the genes CCD1 and CCD4 promoters in vitro.