Modification of pEtc20 with a Modified Cauliflower mosaic virus 35S Promoter

Abstract

            The Cauliflower Mosaic Virus (CaMV) 35S promoter has been the go-to promoter for viral and mRNA work in plants. The promoter was originally mapped in turnip, a dicot. The promoter works well in dicots but does not have as much success in monocots, such as grasses. It is our aim to provide evidence that the CaMV 35S promoter is in need of modification to work in grasses. Prior to our experiment, K. Scheets constructed two Maize chlorotic mottle virus (MCMV) plasmids with CaMV 35S promoters. One plasmid, pCaMCMV, was constructed with a promoter: virus junction according to the published transcription start site of the 35S promoter. It was essentially noninfectious, infecting only 1% of plants inoculated. The second plasmid, pEtc20, was constructed with four additional bases at the beginning of the MCMV sequence. It was much more infectious, infecting 60% of the plants inoculated.  Using this preliminary data, we attempted to assemble two MCMV plasmids with modified promoter/transcription start sites of CaMV 35S. Our initial attempt was unsuccessful, with either pEtc20 being recovered or no DNA recovered at all. New primers with revised melting temperatures were obtained. These were used for another attempt at joining the promoter and MCMV regions. If we are correct, researchers will be able to use the CaMV 35S promoter for better results in mRNA and viral work in monocots.