Structure and promoter/leader deletion analysis of mirabilis mosaic virus (MMV) full-length transcript promoter in transgenic plants.

Abstract

A full-length transcript (FLt) promoter fragment was isolated from a genomic clone of mirabilis mosaic virus (MMV), a double-stranded DNA plant pararetrovirus belonging to the caulimovirus family. The boundaries required for maximal promoter expression were defined by 5' and 3' deletion analysis of the MMV promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated both in transgenic Nicotiana tabacum cv. Samsun NN plants and in protoplast transient expression experiments. A 360 bp FLt promoter fragment (sequence -297 to +63 from the transcription start site) was found sufficient for strong promoter activity. The transcription start site (TSS) of the MMV FLt promoter was determined by primer extension analysis using total RNA isolated from transgenic plants containing a MMV promoter:uidA fusion gene. Analysis of the 5' and 3' deletion constructs showed that an upstream region (sequence -248 to -193 from the transcription start site) is required for the MMV FLt promoter activity along with the as-1, TATA box regions. In addition, a 31 bp sequence (+33 to +63 from the transcription start site) located downstream of a TATA box is also essential for the maximum expression of the MMV FLt promoter. Analysis of transcripts (mRNA) from these chimeric constructs also indicated that the MMV FLt promoter fragment (-297 to +63 from the transcription start site) has the highest promoter activity. In a comparative analysis the MMV FLt promoter showed much greater activity than the CaMV 35S promoter.