Final report SRDC Project QT002 Development of transformation cassettes for sugarcane
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There are currently few gene regulation sequences (promoters/terminators) which have been shown to be effective in sugarcane. At present, the industry standard gene regulatory sequences used for genetic engineering of sugarcane are the maize polyubiquitin (ubi) promoter and the nopaline synthase ( nos) terminator. The overall aim of this project was to develop further promoter and terminator sequences for use in the generation of transgenic sugarcane by testing whether the regulatory sequences derived from banana bunchy top nanovirus (BBTV) would function in sugarcane. Initially, a number of different native BBTV promoters were assessed for expression of the green fluorescent protein (GFP) reporter gene in transient assays, and several of the BBTV promoters were found to express GFP as well as the ubi control. However, when stably transformed mature sugarcane plants were analysed, expression of GFP was almost always abolished. In an attempt to enhance expression, the BBTV promoter sequences were modified by the inclusion ofthe intron from the ubi promoter. In transient assays, the BBTV promoters performed as well as, or better than, the ubi control. When stably transformed plants were analysed by western blot, GFP expression levels equal to that of the ubi control were observed. These results indicated that (i) the inclusion of the ubi intron enhanced the activity of the BBTV-derived promoters and (ii) the promoter sequences derived from BB TV could be used to drive trans gene expression in sugarcane. The BBTV promoters were assessed for their ability to drive the selectable marker gene, neomycin phosphotransferase (nptII). Initially, several native BBTV promoters were tested but none of the transformed sugarcane callus survived selection, indicating that these promoters were unable to drive sufficient levels of nptII expression. When several intron-enhanced promoters were tested, transformed callus was able to survive the selection process, but plantlets could not be regenerated due to problems with the callus. Although preliminary, these results indicated that intron-enhanced promoter sequences from BBTV could be used to drive the nptH selectable marker gene in sugarcane. Several BBTV terminator sequences were assessed for their ability to terminate transcription in sugarcane using nos as a control. These studies revealed that the BBTV termination sequences were as effective as nos in terminating transcription in both transiently and stably transformed sugarcane, thus providing alternative termination sequences for the industry. Constructs containing the promoters from BBTV DNA-4, 5 or 6, the ubi intron and the terminator from BBTV DNA-6 have been prepared. These constructs also contain a multiple cloning site to facilitate use in a variety of systems in sugarcane. Further, two cassettes for selection of transformed tissue have also been prepared which contain the promoter from either BBTV DNA-6 or -4, the ubi intron, the NPTII selectable marker gene and the terminator from BBTV DNA-3.