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 color 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 Msp I/ Hpa II 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. MOLECULAR GENETICS* The text was submitted by the author in English.

An ActiveAc/DsTransposon System for Activation Tagging in Tomato Cultivar M82 Using Clonal Propagation

“…The potential influence of these elements is consistent with the clear tendency for frequent germinal transposition of Ds to occur in T0 families where an initial transposition event had already taken place. These putative interactions between genomic elements and transposase expression are supported by the current body of evidence concerning the influence of genomic elements on gene expression within T-DNA insertions (Mirza, 2005;Filipecki and Malepszy, 2006). Classification of Ds insertions by their flanking regions demonstrated insertion site preferences reasonably consistent with results from screens of over 10,000 tagged lines in Arabidopsis, favoring insertion into genes and euchromatin (Kuromori et al, 2004).…”