Molecular genetic analysis of silencing in two lines ofTriticum aestivumtransformed with the reporter gene construct pAHC25.

Howarth, Jonathan R.; Jacquet, Jean N.; Doherty, Angela; Jones, Huw D.; Cannell, Martin E.

Unpredictable silencing of foreign genes introduced into wheat by particle bombardment transformation has been previously observed. To investigate the cause of this silencing, we selected two independent wheat lines that had been transformed via the particle gun with plasmid pAHC25 containing the genesuidA (GUS) andbar(BASTA herbicide-resistance) each controlled by the maize ubiquitin promoterintron (Ubil). One of these lines (WT2) had shown progressive reductions in transgenic phenotype in successive generations until GUS activity and herbicide-resistance were undetectable in all progeny of the T3 generation tested. A second line, (WT1) exhibited the expected levels of GUS expression and herbicide-resistance up to the T3 generation except for the loss of transgenic phenotype in a single T1 segregant. Transgene stability, silencing and integration locus was investigated in three generations in both lines. The silencing observed in the WT2 line was found to be acting at the transcription level for both theuidA andbargenes. Analysis of transgene promoter methylation in selected T1, T2 and T3 individuals revealed a positive link between methylation status and gene silencing. The silenced T1 individual from line WT1 was also linked to promoter methylation and insertion locus rearrangement. Fluorescencein situhybridisation (FISH) revealed highly divergent transgene insertion loci for WT1 and WT2. WT1 was detected close to the telomere whereas the WT2 insertion was located in the region of the centromere. The effect of these findings on transgene silencing and stability are discussed.

METHYLATION; GENE silencing; TRANSGENES; WHEAT; GENES

Annals of Applied Biology, 2005, Vol 146, Issue 3, p311

0003-4746

Academic Journal

10.1111/j.1744-7348.2005.040121.x