Poster: Regulation of gene expression
Abs #
801: Isolation of a full-length transcript promoter from the Strawberry vein banding virus (SVBV) and expression analysis by protoplasts transient assay and transgenic plants
|
|
Presenter: |
Maiti, Indu B., imaiti@uky.edu |
Authors | Maiti, Indu B. (A) Pattanaik, Sitakanta (A) Dey, Nrisingha (A) Bhattacharyya, Somnath (A) | | Affiliations: |
(A): University of Kentucky
|
| Web Site: | http://www.uky.edu/ktrdc | |
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 ß-glucuronidase (GUS) reporter gene. The expression level of the chimeric constructs made from deleted SVBV promoter fragments have been evaluated in a transient protoplast expression assay. A 371-bp promoter fragment (-352 to +19 from the transcription start site; TSS) was found sufficient for maximal promoter activity and was chosen for further analysis. Comparative transient expression analysis in tobacco and maize protoplasts showed that the activity of this promoter was 6-fold higher in tobacco protoplasts compared to maize. 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. A quantitative GUS activity assay showed that in transgenic tobacco plants the average activity of the promoter was about 3-fold higher in roots than in leaves. Real-time quantitative RT-PCR (qRT-PCR) revealed that this higher level of 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.
