Isolation of full-length transcript promoter from the Strawberry vein banding virus (SVBV) and expression analysis by protoplasts transient assays and in transgenic plants

Abstract

A full-length transcript (FLt) promoter was isolated from a genomic clone of Strawberry vein banding virus (SVBV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family, and its activity was analyzed both in protoplasts and transgenic plants. The 5′–3′-boundaries required for maximal promoter activity were determined by 5′- and 3′-end deletion analysis of the SVBV promoter fused to a β-glucuronidase (GUS) reporter gene. A 371-bp promoter fragment (−352 to +19 from the transcription start site; TSS) was found sufficient for maximal promoter activity in a transient protoplast expression assay, and this was chosen for further analysis. Finer deletion analysis of a 90-bp sequence (coordinates −392 to −302 from TSS) of the SVBV FLt promoter revealed the presence of a negative and a positive regulatory element in this region. In gain-of-function experiment, the fusion of the putative positive regulatory elements with minimal promoter showed very little increment in promoter activity, suggesting that a combinatorial action of various cis-sequences is involved in promoter function. The transcription start site of the full-length transcript promoter was mapped to an A-residue that is located 25-bp downstream of the TATA-box. In protoplast transient expression analysis, the SVBV FLt promoter showed about six-fold higher activity in tobacco compared to maize. A quantitative GUS activity assay showed that in transgenic tobacco plants the average promoter activity was about three-fold higher in roots than in leaves, and this higher activity was due to the accumulation of more GUS specific mRNA in roots. Real-time qRT-PCR analysis and quantitative GUS activity assay showed that the relative strength of the SVBV FLt promoter was greater than the CaMV35S promoter in transgenic tobacco plants. The SVBV FLt promoter is a strong, constitutive promoter and has great application potential in expression of foreign genes in plants.