Cloning And Expression Activity Analysis Of The VwTyDCs Gene And Its Promoter In Pansy Tricolo

Plant tyrosine decarboxylase(TyDC),which decarboxylates tyrosine to tyramine,is thought to play an important role in the formation of floral pigments in pansies(Viola ×wittrockiana).To further clarify the possible mechanism of the tyrosine decarboxylation gene(VwTyDC)for pansy flower color formation,we performed a clonal analysis of VwTyDC and found that in addition to the known VwTyDC,a novel Ty DC gene,called VwTyDC-like,exists.We further cloned the g DNA for VwTyDC and VwTyDC-like genes and VwTyDC-like was analyzed bioinformatically.The expression patterns of VwTyDC and VwTyDC-like genes were analyzed by q RT-PCR.The cis-acting elements on the sequences were analyzed,and the promoter expression activity was analyzed using the promoter-GUS reporter system.The main experimental results are as follows.1.The exon-intron structure of VwTyDC and VwTyDC-like both contain two exons and one intron.The coding region of the VwTyDC-like is 1,497 bp and encodes 498 amino acids.VwTyDC-like is presumed to be an unstable hydrophilic protein with 42 phosphorylation sites and no signal peptide or transmembrane structural domain.The molecular weight of predicted protein is 54.94 k Da,the theoretical isoelectric point is 5.87,the instability coefficient is 47.97.Amino acid alignment analysis showed that the amino acid sequence homology between VwTyDC-like and VwTyDC was 92.37%,the homology with Ty DC from other species were66.18%,such as Setaria viridis,Hevea brasiliensis and Populus trichocarpa.Subcellular localization results showed that VwTyDC and VwTyDC-like were localized in the nucleus and cell membrane.2.qRT-PCR analyses indicated the expression patterns of VwTyDC-like and VwTyDC were highly consistent in different tissues,different stages of flower development,different areas of labellum and different flower colors,but the expression level of VwTyDC-like was generally higher than that of VwTyDC.Both VwTyDC-like and VwTyDC had the highest expression levels in pansy flowers.The expression levels of VwTyDC-like and VwTyDC in the central blotch areas were significantly higher than non-blotch areas at stage 7 of flower development.In pansy cultivars with different flower colors,regardless of petals with or without flower blotches,the expression levels of these two genes in the central blotches/their corresponding areas were obviously higher than non-blotches/their corresponding areas.Meanwhile,in different regions of labellum in both blotch and non-blotch cultivars,the expression of both VwTyDC-like and VwTyDC decreased gradually from the proximal of petals outward.Therefore,we speculate that the expression of VwTyDC-like and VwTyDC genes in pansy petals exhibits obvious positional effects.3.The genomic walking by FPNI-PCR and TAIL-PCR was used to successfully clone2,254 bp and 1,201 bp of the 5’ upstream flanking sequences of the transcription start site of VwTyDC-like and VwTyDC.Cis-acting element analysis showed that both VwTyDC-like and VwTyDC contained multiple core initiation elements,TATA-box and CAAT-box.The distribution and number of the core initiation elements showed that both VwTyDC-like and VwTyDC has initiation activity in the 5’ upstream sequences.The promoter regions of VwTyDC-like and VwTyDC contain several identical or similar cis-acting elements,and the promoter regions contain elements that are responsive to light regulation and hormones,etc.The isolated promoter sequences were fused with the reporter GUS to construct a plant expression vector to transform Arabidopsis,and the expression activity of the promoters was detected by GUS histochemical staining,and the results showed that the VwTyDC-like and VwTyDC promoters are flower-specific promoters and the expression activity in petals has a positional effect,and the results of GUS fluorescence quantification indicated that the VwTyDC-like and VwTyDC promoters have high transcriptional efficiency in flowers...