Abstract
BackgroundDevelopment of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications.MethodologyUsing the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter.Significance/ConclusionBoth shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants.
Highlights
In the eukaryotic cell, expression of a transgene depends upon the presence of the primary regulatory element, the promoter, which plays a major role in determining the relative level of transcription and, gene expression and function
We developed a pair of hybrid promoters, viz., FuasFScp and FSuasFcp by intermolecular exchange of the important domains of F and FS promoters
A total number of 300 pUCPMAGUS based shuffled promoter clones (50 shuffled promoter clones each from six different shuffled-promoter libraries), and clones with F, FS and CaMV35S promoters fused to the GUS reporter gene were evaluated in tobacco
Summary
Expression of a transgene depends upon the presence of the primary regulatory element, the promoter, which plays a major role in determining the relative level of transcription and, gene expression and function. The basic rationale behind developing such modified promoters lies in the notion that the transfer of the upstream DNA sequence/cis-element that binds a specific trans-factor from one promoter into a different promoter containing the TATA sequence might result in a novel regulatory or transcription model [12]. Apart from these approaches, linker scanning mutagenesis [13] and error prone PCR [14] have been used to introduce either random or specific mutations into a promoter sequence – the objective being to alter either the orientation/arrangement of the existing cis elements or to insert or destroy cis-elements that modify the existing function of a promoter. Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
