Molecular cloning and in-silico analysis of Ramy3D promoter and 5′ untranslated region from an Iranian rice (Oryza sativa L.) cultivar “NEMAT”

Abstract

The regulatory sequence of rice alpha amylase 3D gene (<em>Ramy3D</em>) is amongst the most successful expression systems used for recombinant protein expression in plants. In the current study a 995 bp fragment consisting of<em> Ramy3D</em> promoter and its 5′ untranslated region was amplified from the genomic DNA of an Iranian rice cultivar ″Nemat″, using polymerase chain reaction. The amplified fragment was ligated into the pTG19-T vector and the cloned fragment was sequenced. For <em>in silico </em>characterization, the rice specific consensus sequences of TATA-box and YR Rule motifs were scanned against the cloned fragment sequence using FIMO program and the cis acting elements existing in the promoter region were investigated using PlantCare database. A TATA-box motif with the rice specific pattern was identified at upstream position of the transcription start site. The identification of TATA-box in <em>Ramy3D</em> promoter is consistent with its metabolic and tissue specific regulation manner. Several cis regulatory motifs responsible for the metabolic and hormonal regulation of <em>Ramy3D</em> gene were identified including ABRE, G-Box, GC-box, GATA motif and TATCCA T/C motif. In addition, several motifs involved in response to various stimuli such as plant hormones, light and biotic and abiotic stresses were identified which include circadian motif, as-2-box, WUN-motif, TGACG-motif, Skn-1 motif, O2-site, MBS, LAMP-element, I-box, HSE, GCC Box, GATT motif, CGTCA-motif and GAG-motif.