Influence of the nature of the T-DNA insertion region on transgene expression in Arabidopsis thaliana

Abstract

In the experiment reported here, effect of the nature of T-DNA integration region on the activity of the transgenes was studied by using a colour marker gene in Arabidopsis thaliana. For this purpose a pale homozygous ch-42 mutant was transformed with the wild-type copy of the gene (CH-42) using kanamycin resistance gene as a selectable marker. Two independent lines were identified in which CH-42 transgene was inactive. The T-DNA flanking sequences were recovered from these inactive and two active lines. These flanking sequences were used to examine copy number and DNA methylation of the T-DNA insertion site in active and inactive lines. Southern blots produced by using MspI/HpaII digested genomic DNA showed signs of methylation in both inactive lines. Furthermore, in one of the inactive line the T-DNA flanking sequence probe hybridized to highly repetitive sequence. The results suggest some correlation between silencing of the transgene and methylation of its insertion region.