title: Novel Synthetic Promoters from the Cestrum Yellow Leaf Curling Virus.
abstract: Constitutive promoters direct gene expression uniformly in most tissues and cells at all stages of plant growth and development; they confer steady levels of transgene expression in plant cells and hence their demand is high in plant biology. The gene silencing due to promoter homology can be avoided by either using diverse promoters isolated from different plant and viral genomes or by designing synthetic promoters. The aim of this chapter was to describe the basic protocols needed to develop and analyze novel, synthetic, nearly constitutive promoters from Cestrum yellow leaf curling virus (CmYLCV) through promoter/leader deletion and activating cis-sequence analysis. We also describe the methods to evaluate the strength of the promoters efficiently in various transient expression systems like agroinfiltration assay, gene-gun method, and assay in tobacco protoplasts. Besides, the detailed methods for developing transgenic plants (tobacco and Arabidopsis) for evaluation of the promoter using the GUS reporter gene are also described. The detailed procedure for electrophoretic mobility shift assay (EMSA) coupled with super-shift EMSA analysis are also described for showing the binding of tobacco transcription factor, TGA1a to cis-elements in the CmYLCV distal promoter region.
authors.affiliation: KTRDC, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, USA.|Department of Agronomy, Iowa State University, Ames, IA, 50011, USA.
authors.forename: Dipak Kumar
authors.initials: DK
authors.lastname: Sahoo
authors.affiliation: Department of Gene Function and Regulation, Institute of Life Sciences, Chandrasekharpur, Bhubaneswar, Odisha, India.
authors.forename: Shayan
authors.initials: S
authors.lastname: Sarkar
authors.affiliation: KTRDC, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, USA.
authors.forename: Indu B
authors.initials: IB
authors.lastname: Maiti
authors.affiliation: Department of Gene Function and Regulation, Institute of Life Sciences, Chandrasekharpur, Bhubaneswar, Odisha, India. nrisinghad@gmail.com.|Department of Biotechnology, Government of India, Chandrasekharpur, Bhubaneswar, Odisha, India. nrisinghad@gmail.com.
authors.forename: Nrisingha
authors.initials: N
authors.lastname: Dey
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000821:virology; D018506:Gene Expression Regulation, Plant; D016679:Genome, Viral; D008967:Molecular Biology / Q000379:methods*; D010942:Plant Viruses / Q000235:genetics* / Q000472:pathogenicity; D030821:Plants, Genetically Modified / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000821:virology
keywords: Arabidopsis; Caulimovirus; GUS; Gene expression; Synthetic promoter; Tobacco; Transgenic plants