Analysis of Dahlia Mosaic Virus Full-length Transcript Promoter-Driven Gene Expression in Transgenic Plants
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A 556-bp long full-length transcript promoter (DaMVFLt4-; −474 to +82 from transcription start site) with enhanced activity was characterized from the Dahlia mosaic virus (DaMV). The strength of the DaMVFLt4- promoter has been evaluated in transient systems and in transgenic plants using two different reporter genes like β-glucuronidase (GUS) and green fluorescent protein (GFP). The DaMVFLt4- promoter was found to be 4-fold and 5-fold stronger than the CaMV35S promoter in tobacco protoplast and transgenic tobacco plants, respectively. Electrophoretic mobility shift assay (EMSA) and supershift analysis confirmed the binding of tobacco transcription factor TGA1a to the enhancer region of the DaMVFLt4- promoter. TGA1a specially interacted with the activation sequence-1 (as-1) (−69 to –41 from transcription start site (TSS)) of DaMVFLt4- promoter as shown by DNaseI footprinting. UV cross-linking studies and Southwestern blot analysis clearly demonstrated that the purified TGA1a specifically bound to as-1 element, whereas in tobacco nuclear extract, two unknown transcription factors of about 41 kDa (putative TGA1a) and about 67 kDa were bound to the as-1 sequence of the DaMVFLt4- promoter. Expression studies with the DaMVFLt4-::β-glucuronidase (GUS) genes in tobacco protoplasts co-transfected with CaMV35S::TGA1a showed that expression of TGA1a resulted in approximately 3.7 times elevated levels of GUS activity. The DaMVFLt4- promoter is a constitutive promoter, and the expression level in tissues of transgenic tobacco plants was in the order root > leaf > stem. In addition, the DaMVFLt4- promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived DaMVFLt4- promoter would become an efficient tool for biotechnological application.
KeywordsDNA Promoter Caulimovirus Transgenic plants GUS GFP Confocal microscopy
We are very much grateful to the Kentucky Tobacco Research and Development Center (KTRDC) for the facilities and support. This work was supported by the KY state KTRDC grant to IBM. The authors would like to thank Ms. Bonnie Kinney for her excellent care of the experimental tobacco plants, Mr. Abhimanyu Das, ILS for the technical assistance, and Dr. Sitakanta Pattanaik, University of Kentucky for the suggestions and technical advice.
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