Silenziamento genico in piante di vite trasformate per la resistenza a Grapevine fanleaf virus.

In grapevines previously obtained, produced via Agrobacterium-mediated transformation and containing the coat protein gene of Grapevine fanleaf virus (GFLV-CP), we did not observe a strict correlation between number of T-DNA insertions and mRNA accumulation levels. To gain insight into T-DNA integration process in grapevine, 49 transgenic grapevine lines were analyzed. Sequence comparison revealed several deletions in the T-DNA and duplications or deletions of grapevine DNA at the T-DNA insertion loci. Multiple T-DNAs frequently integrated at the same position in the plant genome, resulting in the formation of tandem or inverted repeats. Eight transgenic grapevine lines were further analyzed for a correlation among transgene expression, siRNAs production and DNA methylation. Highly methylated cytosine residues were detected in the transgene sequence of three grapevines without transgene expression, but no detectable level of siRNAs was recorded in these lines. Complex arrangements of T-DNA and integrated binary vector sequences appear to be crucial factors that can influence transgene expression.

Piante di vite trasformate con il gene della proteina capsidica del Grapevine fanleaf virus (GFLV-CP) non presentano una correlazione diretta tra espressione e numero di copie dei transgeni inseriti. Analisi molecolari hanno evidenziato delezioni e duplicazioni del T-DNA e del DNA di vite nei punti di inserzione del transgene. In alcune linee in cui più copie di T-DNA sono state integrate in un singolo locus l'espressione del transgene è in genere molto bassa o assente. Nelle linee silenziate sono state osservate elevate percentuali di citosine metilate a livello del transgene, ma non sono stati individuati siRNAs specifici per GFLV-CP.