Transgene silencing in wheat (Triticum aestivum) transformed with the wheat streak mosaic virus coat protein (WSMV-CP) gene

Wheat (Triticum aestivum) was transformed with a Wheat streak mosaic virus coat protein (WSMV-CP) gene by the biolistic method. The transgene product, WSMV coat protein, was necessary for WSMV resistance in transformed wheat. However, transgene silencing was frequently observed in wheat containing WSMV-CP genes and caused unpredictable segregation of the introduced trait in successive generations. In this report, a transgene silencing line, 566B, was studied in detail. Seedlings from selfed generations of 566B, from T₁ to T₃, were screened for presence and expression of the WSMV-CP gene by polymerase chain reaction (PCR), Enzyme-linked Immunosorbent Assay (ELISA), Southern blot and western blot analysis. The results indicated that all the transgenic 566B T₁ plants containing WSMV-CP gene can express WSMV coat protein and had strong resistance to WSMV. While the WSMV-CP transgene is stably maintained through the T₂ and T ₃ generations, transgenic plants in these two generations showed transgene silencing and were susceptible to WSMV infection.; Reversal of transgene silencing using the cytidine analog, demethylating agent 5-azacytidine (5-AzaC) has been reported in plants, but this phenomenon had not been reported in transgenic wheat. In this research, transgene silencing of 566B plants could be reversed by 5-AzaC treatment, and upon reversal these plants were resistant to WSMV. However, the effect of 5-AzaC on transgene expression was temporary and transgene silencing was re-established 15 to 20 days after the treated plants were transplanted into soil in the absence of 5-AzaC. This provided direct evidence that DNA methylation was involved in the transgene silencing, and indicated that the silencing was at the transcriptional level. Several possible mechanisms of transgene methylation and inheritance such as “global methylation events of normal plant development”, “DNA repeats”, and “DNA methylation spread” are discussed.; A quantitative real-time PCR technique, in which a wheat housekeeping gene, puroindolin-b, was used as an internal control, was successfully developed to determine transgene copy number in transgenic wheat. By comparison between “estimated” copy number in transgenic lines using real-time quantitative PCR and ‘actual’ copy number based on Southern blot analysis, real-time PCR was 80–90% in agreement with Southern blot results. (Abstract shortened by UMI.)...