Isolation, cloning, and functional analysis of a putative constitutive promoter of E3 ubiquitin‐protein ligase RF4 from Coleus amboinicus Lour

A promoter is a region in the genome sequence located upstream of the transcription start site comprising cis acting elements that initiates and regulates the transcription of an associated genes and restriction endonucleases. As the need for genetically engineered plants has widened, the requirement to develop methods to optimize the control of transgene expression has also increased. Therefore, analyzing the functionality of the promoter is very important in understanding the target gene expression. The widespread use of viral constitutive promoters (cauliflower mosaic virus, CaMV35) has raised concerns about the safety and containment of transgene in the environment. Hence isolation and characterization of novel promoters using fast and efficient genetic engineering tools is the need of the hour. The present study, for the first time, describes the isolation and characterization of a novel constitutive promoter driving ubiquitin E3 ligase from the plant Coleus amboinicus, a perennial herb, of the Lamiaceae family. The functionality of the isolated promoter was demonstrated using the β ‐glucuronidase as a reporter in tobacco var Petit havana. The development of blue color in the tobacco leaves indicated the presence of a functional promoter. Graphical : The study was aimed to isolate the putative promoter of constitutively expressing the E3 ubiquitin ‐protein ligase RF4 gene, of the ubiquitin‐proteasome system from Coleus amboinicus Lour using Thermal Asymmetric InterLaced PCR (TAIL PCR) method. The isolated putative promoter was subjected to in silico analysis for the presence of the minimal promoter elements. The functional analysis of the promoter was done by transforming tobacco var Petit Havana followed by histochemical localization using the GUS reporter gene.