title: Development of useful recombinant promoter and its expression analysis in different plant cells using confocal laser scanning microscopy.
abstract: BACKGROUND
  Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy.
  
  
  RESULTS
  We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared.
  
  
  CONCLUSIONS
  We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety of plant cells.
authors.affiliation: Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India, Nalco Square, Chandrasekherpur, Bhubaneswar, Orissa, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Kumar
authors.forename: Sunita
authors.initials: S
authors.lastname: Patro
authors.forename: Rajiv
authors.initials: R
authors.lastname: Ranjan
authors.forename: Dipak K
authors.initials: DK
authors.lastname: Sahoo
authors.forename: Indu B
authors.initials: IB
authors.lastname: Maiti
authors.forename: Nrisingha
authors.initials: N
authors.lastname: Dey
mesh_terms: D018613:Microscopy, Confocal / Q000379:methods*; D059828:Plant Cells / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism