Evaluation of the promoter activity of caulimoviruses associated with Dahlia spp by transient expression of the beta-glucoronidase gene (GUS)

Funding agencies: Samuel and Patricia Smith Endowment for Dahlia Virus Research created by the American Dahlia Society, the USDA Northwest Nursery Crop Research Center and Washington State Commission for Pesticide Registration.One of the most common promoters used for constitutive gene expression was derived from a plant virus. The 35S promoter from Cauliflower mosaic virus (CaMV) drives high levels of transgene expression; directs constitutive expression and displays no tissue-specificity. Due to these properties, the CaMV 35S promoter is widely applied in expressing foreign genes in plants. However, some limitations including insufficient promoter activity or strong down-regulation necessitated the exploration of more efficient promoters from other caulimoviruses. Dahlia mosaic virus (DMV) and two new caulimoviruses tentatively designated as Dahlia common mosaic virus (DCMV) and an endogenous sequence (DMV-D10) have been reported from dahlia (Dahlia variabilis) plants. Not much information is known about the promoters from these caulimoviruses and they represent a potential source for new promoters. Based on sequence comparisons and promoter prediction programs, we identified the putative 35S promoter in DMV, DCMV and DMV-10 from cultivated and wild dahlia species. The intergenic regions containing the putative 35S promoter were separately cloned into pCAMBIA1281Z, a binary vector. Constructs were delivered into Agrobacterium tumefaciens by electroporation and agroinfiltrations were done into Nicotiana tabacum, N. benthamiana and Verbesina encelioides. The activity and strength of the putative promoters from DMV, DCMV and DMV-D10 was determined by GUS assays. Preliminary results from qualitative GUS assays demonstrated that DMV, DCMV and DMV-D10 promoter activity is similar to the one showed by 35S promoter of CaMV. Transient expression showed stronger activity in N. benthamiana leaf tissue in comparison to N. tabacum leaf tissue for all constructs. Deletion analysis of the 3' and 5' end of the promoter region in order to establish the optimal boundaries for maximal promoter activity is underway. Quantitative GUS assays are also in progress. Since promoter selection has become increasingly important for successful gene transfer and expression of transgenes in plants, the findings could provide important information on new promoters for gene expression in plants.Washington State UniversityAlmeyda, J. L. et al. (2010, March 26). Evaluation of the promoter activity of caulimoviruses associated with Dahlia spp by transient expression of the beta-glucoronidase gene (GUS). Poster presented at the Washington State University Academic Showcase, Pullman, WA