Special attention needs to be given to defining and studying the regulatory apparatus of different pararetroviral promoters under various physiological conditions because they have significant sequence heterogeneity and unique distributions of stress-responsive cis-elements. Transcriptional regulation studies of a pararetroviral promoter involve both gene expression analyses and investigation of its structural/regulatory framework. The expression of reporter genes such as β-Glucuronidase (GUS) or Luciferase (LUC) transcriptionally fused to a promoter usually determines the strength or function of a target promoter. In parallel, DNA-protein interaction studies are employed to assess the functional relevance of predicted transcription factor binding sites in target pararetroviral promoter sequences. In this chapter, we will describe protocols used to determine the transgene integration and expression in transgenic plant systems. Alongside, we will also discuss the fusion reporter assays that can determine the promoter activity and DNA-protein interaction studies that aid in the evaluation of its transcriptional regulation.
Keywords: EMSA; GUS; Histochemical; Luciferase; Real-time PCR.