T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange

Abstract

In transgenic plants, multiple T-DNA copies and cytosine methylation usually correlate with a loss of transgene expression while methylation of promoter is associated with transgene silencing. Here, six independent GFP transgenic ‘Bingtang’ sweet orange (Citrus sinensis (L.) Osb.) plants were analyzed with copy number, T-DNA repeat, cytosine methylation and transgene expression. The fluorescence of transgenic plants was normal and bright except that the transgenic B4 plant showed mottled fluorescence. Five of the six transgenic plants had multiple transgene copies and three transgenic plants had direct repeat sequences. The degrees of cytosine methylation within GFP and CaMV 35S promoter region were higher in multiple copies than in single copy transgenic plant B6. Cytosine methylation of nptII in multiple copies transgenic plants except B4 was slightly greater than single copy transgenic plant B6. Furthermore, transgenic B4 plant with mottled GFP expression had the highest degree of methylation of CG cytosine pattern. The transcript level of GFP and nptII was lower in three transgenic plants (B1, B2 and B3) with direct repeat, as revealed by real-time RT–PCR analysis. These results suggest that multiple T-DNA copies and methylation of CaMV 35S promoter affect transgene expression levels but do not cause gene silencing in transgenic sweet orange.