Tandem repeat sequences in a transcriptional unit cause high -frequency transgene silencing in primary transgenic tobacco

As transformation becomes a routine for many plant species, expression of multiple transgenes in a plant is expected to be in great demanding. Traditionally, mufti-genes are transferred separately and are brought together later by progressive crossings. The requirements of a large amount of labor and time make this strategy inefficient. In this study, a new and simple strategy was established using the autoproteolytic 2A protease of the foot-and-mouth disease (FMDV) as a linker to fuse mufti-genes into a single open reading frame (ORF). The 2A protease will cotranslationally process the polyprotein in an autocleavage way, thus releasing individual protein of the multiple genes from the polyprotein. Using this strategy, up to 4 gene fragments were expressed in a single transformation procedure although the processing efficiency of the 2A protease was about 80%. However, when we tried to link 2 to 4 repeated GUS or CAT gene into an ORF, severe gene silencing was found in 80–100% primary transgenic plants carrying a construct with repeated sequences. Further analyses revealed that this phenomenon might represent a universal mechanism since all three genes, CAT, GUS and GFP, were sensitive to the silencing mechanism when repeated sequences of a gene were incorporated into a single transcriptional unit. In the cases where transgenic plants contained a construct with 3 or 4 tandemly-repeated CAT gene fragments, the silencing was independent of T-DNA copy number and was often associated with heavy methylation in the coding region. The silenced state in plants containing a construct with 3 or 4 repeats could be stably inherited, but reversion of silencing often occurred in plants carrying a construct with 2 repeats, suggesting a possible gene dosage effect. Although the precise mechanism of this silencing phenomenon is undetermined, failure to reactivate the silenced genes with 5-azacytidine treatment might indicate the involvement of a posttranscriptional gene silencing mechanism. We also investigate the potential utilities of this phenomenon in inactivation of endogenous genes. Results showed promising