Digitalis türlerinden izole edilecek 3 beta HSD ve 5 beta POR genlerine ait promotorların Arabidopsis thaliana'da analizi

Digitalis ferruginea, known as foxglove, is famous for the production of secondary metabolites; i.e., cardiac glycosides. The progesterone 5β-reductase (5βPOR1 and 5βPOR2) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are important key step genes in the pathway of biosynthesis of cardenolide in Digitalis species. For the first time, promoters of 3β-HSD, 5βPOR1 and 5β POR2 were isolated from genomic DNA of Digitalis ferruginea and sequence analysis was performed. Sequence information of the promoters revealed that these promoters have abundant cis-elements related to light responses. 5βPOR1promoter specific cis-elements are circadian and heat shock. 5βPOR2promoter specific regulatory elements are defense, stress and methyl jasmonate. Wound response element was found specifically in the 3β-HSDpromoter. Functional characterization of the 3β-HSDpromoter::GUS in Arabidopsis plants transformed by floral dip transformation showed activity in cotyledons, newly emerged leaves, vascular tissues of shoot and root, trichomes, shoot apical meristem (SAM) and hydathodes during plant growth and developmental stages. Under the treatments of 3% sucrose or 2% mannitol intensively enhanced GUS activity in the seedlings. In the presence of NaCl (150 mM) promoter expression activity was downregulated. 5βPOR1promoter::GUS activity was the highest in 5 and 10 days old seedlings in all tissues except root apical meristem, then decreased in 15 days old seedlings. 5βPOR2promoter::GUS activity was confined to vascular tissues in cotyledons in 5 and 15 days old seedlings. In 10 days old seedlings, promoter activity was intensively found in cotyledons, vascular tissues of cotyledons, hypocotyl and root adjacent to hypocotyl. Promoter activity was intensive in hypocotyl of 15 days old seedlings. The promoter sequences and functional characterization in Arabidopsis, revealed that the genes are light regulated and the 3β-HSD have role in plant growth, development and abiotic stress tolerance. Further, the genes 5βPOR1 and 5βPOR2 also have crucial role in normal plant growth during developmental stages.