Transposon Tn5 mediated gene transfer into plants

Abstract

A novel bacterial transposon, Tn5-PV, that can used for mapping and analysis of the expression of genes transferred into plant cells, was constructed by insertion of a plant gene vector into Tn5 without alteration of its transposition properties. Tn5-PV carries ori V and ori T sequences of the broad host range plasmid RK2 linked to selectable and screenable plant marker genes and to the 25 bp border sequences of Agrobacterium Ti plasmid T-DNAs, orientated towards the ends (IS50L and IS50R) of Tn5. Plasmids with Tn5-PV insertions are converted by the transposon insertion into plant gene vectors that can be efficiently mobilized from Escherichia coli and stably maintained in Agrobacterium hosts. Due to the orientation of the T-DNA borders. Tn5-PV mediates the transfer of target plasmids from Agrobacterium to plant cells. The usefulness of this approach was demonstrated by transferring independent transposon insertions in T-DNA tumour genes to tobacco cells. The correlation between the map position of transposon insertions, tumour phenotypes, the absence of specific T-DNA transcripts and the structural analysis of pGV354::Tn5-PV derivatives in DNA from tobacco tumours, showed that T-DNA genes linked to the chimeric plant marker genes of Tn5-PV were transferred without any rearrangement into plant cells.