1Department of Molecular Biology and Genetic Engineering, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, 263 145, India
2National Research Centre on Plant Biotechnology, IARI, New Delhi, 110012, India
3Institue of Microbial Technology (CSIR), Sector 39-A, Chandigarh, 160036, India
4Centre for Bioinformatics, School of Biotechnology, Banaras Hindu University, Varanasi, Uttar Pradesh-221 005, India
5Division of Seed Science and Technology, Indian Agriculture Research Institute, New Delhi, 110012, India
6Department of Biotechnology, D.DU Gorakhpur University, Gorakhpur, Uttar Pradesh-273 009, India
*Corresponding author: dinesh_yad@rediffmail.com
Online published on 4 November, 2016.
The Dof (DNAbinding with one finger) family is a plant specific transcription factor known to be involved with regulating diverse functions in plants and have been extensively studied in many crops. The Dof transcription factor regulating gene expression by interacting with Cis-regulatory elements namely prolamin box (P box), GCN4, AACA and ACGT motifs present in the promoters of seed storage protein genes is known as PBF (Prolamin-box Binding Factor) Dof transcription factor. A set of 15 primers were designed by considering approximately 1.5 kb upstream and 500bp downstream sequences of full length Dof genes from TSS of cereals like rice, wheat and sorghum available in databases. These primers were used for PCR amplification of putative promoters of Dof genes of finger millet along with few cereals and millets. Furthermore, based on the presence of expected size amplicon with different sets of primers tested, a total of 6 bands of expected size representing putative promoters of PBF-Dof genes of rice, sorghum, barnyard, finger millets (PRM-1 PRM-801, PRM-701) were eluted, sequenced and subjected to in silico investigation. The bioinformatics based characterization revealed uniform presence TSS and numerous seed storage protein specific motifs like DPBF motif, RY element, SKN1 motif, GCN4 motif, E-Box confirming the promoters of respective PBF-Dof genes of cereals and millets. Further, validation by cloning in promoter probe vector is required for confirmation of temporal and spatial expression associated with seed storage protein genes.
Primer were designed from rice, wheat and sorghum Dof gene promoters and PCR was attempted in finger millet, rice and sorghum.
In silico analysis of cloned sequences revealed TSS and numerous seed storage protein specific motifs
PRM-1, PRM-701 and PRM-801 showed presence of three DNA motifs which are binding sites for various well characterized transcription factors e.g. MYB, abi4, SOC, FLC, PBF, Dof 2 etc.
Presence of these conserved motifs identifies cloned sequences as putative Dof promoter candidates.
Cereals, millets, prolamin-box binding factor (PBF), Dof (DNA binding with one finger) transcription factor, In-silico, promoters
