Characterization Of OfWRKY3,a Transcription Factor That Positively Regulates The Carotenoid Cleavage Dioxygenase Gene OfCCD4,in Osmanthus Fragrans

Osmanthus fragrans is known as Osmanthus and part of Oleaceae osmanthus,originated from China and cultivated a long history for more than two thousand years.O.fragrans is a famous ornamental plant.O.fragrans is mainly classified to four cultivar groups,respectively,Siji group,Albus group,Luteus group and Auranticus group.Previous studies have proved that the color of Osmanthus petal were much affacted by carotenoid accumulation.The higher carotenoid content in petals,the deeper color will be.The carotenoid cleavage dioxygenase(CCD4)in O.fragrans can splitting carotenoid to produce aromatic ionone.Previous studies have shown that ionones content is the highest composition of essential oil in the O.fragrans petals.Therefore,OfCCD4 gene is the key gene of sweet scented of O.fragrans flowers and flower color decision.Our purpose is to verify of whether the OfWRKY3 transcription factor can regulate OfCCD4 gene and promote its expression,according to the experiments,and study the mechanism of sweet osmanthus flower color and fragrance from the level of molecular biology,this will enrich the basic theory about take advantage of sweet osmanthus,sweet osmanthus fragrance,provide a theoretical basis for the improvement of color and new varieties of sweet scented osmanthus.The main results are as follows:1.Expression patterns of OfWRKY3 and OfCCD4 in different flowering stages of O.fragransTo extract total RNA from O.fragrans petal with Dangui and Yingui in different period,then synthesis by reverse transcript to cDNA,and then through the polymerase chain reaction method to observe the expression of gene WRKY3 and CCD4 in different period.With the gene expression pattern of plant hormone treatment blossom petals after using quantitative PCR method to detect above two genes.2.Subcellular localization of OfWRKY3 in tobacco leavesTo construct 35S-OfWRKY3-GFP vector.The resulting vector was transformed into Agrobacterium tumefaciens strain,using PEG4000 mediated by micro pressure injection way into protoplasts of tobacco leaves,and incubated with 12 h in darkness environment,then were observed through a fluorescence microscope.The results showed that OfWRKY3 protein located in the nucleus.3.The activity of GUS protein was detected in the co-infiltrated tobacco leavesTo construct 35S:OfWRKY3 expression vector and the target plasmid containing OfCCD4 promoter respectively were cotransformed into tobacco leaves via micro pressure injection.After incubated in 36 h,the expression level of GUS staining in tobacco leaves was detected.The results showed that GUS protein was over expressed.4.Transient over expression of OfWRKY3 protein in petals of O.fragransWe constructed 35S:OfWRKY3 expression vector and then transformed into O.fragrans full stage flower petals by vacuum filtration way,and incubated for 36 h in the darkness environment.The total RNA of the petals was exacted for the detection of target gene transcription level by using fluorescence quantitative PCR.The results showed that the transcription level of OfCCD4 gene was significantly affected by the overexpression of OfWRKY3 transcription factors.5 In vitro expression and purification of OfWRKY3 protein.Transcription factor OfWRKY3 gene sequence obtained preliminary amplified with PCR,fragment expression vector was constructed for transcript into E.coli BL21 competent cells.The expression of OfWRKY3 protein was induced by IPTG.The target protein was detected by SDS-polyacrylamide gel electrophoresis,and the target protein purified by Ni-NTA agarose gel purification.6.Eectrophoretic mobility shift assay(EMSA)verify that the OfWRKY transcription factor can bind to the W-box sequence upstream of the OfCCD4 gene.The total O.fragrans DNA was extracted by DNA extract kit,OfCCD4 gene promoter sequence was cloned by PCR,which contains W-box binding sites,and ligated into a pET-30a-c vector.The purified OfWRKY3 was used for EMSA,according to strip mobility shift to determine the OfCCD4 promoter can positively combine with OfWRKY3 transcription factor.DNA binding ability of the protein was thus detected.