PCR amplification and bioinformatics assessment of promoters of PBF-DOF (DNA binding with one finger) genes of finger millet

Abstract

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.