Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2007
Publication Date: 7/25/2007
Citation: Cazzonelli, C., Velten, J.P. 2007. Synthetic plan promoters as a tool for characterizing the function of individual promoter elements. Transgenic Research [serial online]. Available: http://www.springerlink.com/content/w969356312625861/. Interpretive Summary: Synthetic gene regulatory sequences (promoters) were constructed using DNA sequences derived from plant viruses, joined to a luciferase reporter gene and tested for function within living plant tissues. Most DNA regulatory elements (transcriptional enhancers) were found to be additive in function. Some elements also produced promoters that respond to the presence of bacteria within leaves by changing reporter gene expression levels. Data reported in this paper can be used to better design synthetic plant promoters targeted to specific tissues or environmental conditions.
Technical Abstract: Short direct-repeat (DR) DNA enhancer elements from plant viral intergenic regions were analyzed for their ability, both individually and in combination, to influence transcription when inserted upstream from a minimal CaMV35S promoter. Synthetic promoters containing multiple copies and/or combinations of DR cassettes were tested for their effect upon reporter gene (luciferase) expression using an Agrobacteria tumefaciens-infiltration transient assay (Nicotiana tabacum SR1 leaves) and within stably transformed plants. Transgenic plants harboring constructs containing different numbers or combinations of DR cassettes were further tested to look for tissue-specific expression patterns and potential promoter response to the infiltrations process. Multimerization of DR elements produced enhancer activity that was in general additive, increasing reporter activity in direct proportion to the number of DR cassettes within the test promoter. In contrast, combinations of different DR cassettes often functioned synergistically, producing reporter enhancement markedly greater then the sum of the combined DR activities. Several of the DR constructs responded to Agrobacteria (lacking T-DNA) infiltration of transgenic leaves by an induction (2 elements) or reduction (1 element) in reporter activity. Combinations of DR cassettes producing the strongest enhancement of reporter activity were used to create two synthetic promoters (SynPro3 and SynPro5) that drive leaf reporter activities at levels comparable to the CaMV35S promoter. Characterization of these synthetic promoters in transformed tobacco showed strong reporter expression at all stages of development and in most tissues. The arrangement of DR elements within SynPro3 and SynPro5 appears to play a role in defining tissue-specificity of expression and/or Agrobacteria responsiveness.