Promoter deletion and comparative expression analysis of the Mirabilis mosaic caulimovirus (MMV) sub-genomic transcript (Sgt) promoter in transgenic plants
Nrisingha Dey and Indu B. Maiti
A sub-genomic transcript (Sgt) promoter was isolated from a genomic clone of the Mirabilis mosaic virus (MMV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family. The MMV Sgt promoter fragment (genomic co-ordinates 4830 to 5840) was mapped by 5¢-3¢-deletion analysis to define the boundaries required for maximal promoter expression. Expression patterns of promoter fragments coupled to GUS reporter gene were evaluated both in protoplast transient expression experiments and in transgenic Nicotiana tabacum cv. Samsun NN plants. A 333 bp MMV Sgt promoter fragment (sequence –306 to +27 from the transcription start site, TSS) was found sufficient for strong promoter activity in protoplast transient expression experiments. The transcription start site (TSS) of the MMV Sgt promoter was determined by primer extension analysis using total RNA isolated from transgenic plants containing a MMV Sgt promoter:uidA fusion gene. In seedlings, the level of GUS expression was in the order of leaf > root > stem. Histochemical GUS-staining of seedlings showed highest GUS activity in root tips, leaf midribs, veins and vascular tissues. The MMV Sgt promoter fragment is a strong constitutive promoter, with strength comparable to that of the MMV full-length transcript (FLt) promoter. MMV Sgt promoter also demonstrated much greater activity, 8-fold more compared to the CaMV 19S promoter and 2-fold stronger than the CaMV 35S promoter.