Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum

Abstract

Transgenic plants produced by Agrobacterium-mediated transformation usually have one or a few stable and intact T-DNA insertions. However, in a significant number of the transformants Southern blot analysis has revealed the occurrence of aberrant T-DNA insertions missing one or both ends. During the study of this phenomenon, we obtained Km R Nicotiana tabacum clones after cocultivation with an Agrobacterium strain containing a promoterless nptII gene located internally in the T-DNA. Expression of this nptII gene requires a break in the T-DNA region upstream from the nptII-coding sequence and insertion of the truncated T-DNA in a transcriptionally active plant DNA region. The most conspicuous results from Southern analyses on four such Km R plant clones is that they contain several T-DNAs truncated at other positions besides the upstream region of the nptII sequence. Four truncated T-DNA insertions have been cloned. Two insertions contain the nptII gene fused to plant expression signals and are missing the right part of the T-DNA. Another is missing the left T-DNA part and the last T-DNA is lacking both ends. Sequence analysis of the T-DNA:: plant junctions has shown that the T-DNA breakpoints are randomly distributed and do not show obvious homologies to one another or to the border consensus sequence. S1-type mapping of the most strongly expressed plant genome:: nptII fusion revealed a specific transcription start point and putative TATA and CAAT boxes in the upstream plant DNA region; the steady-state nptII mRNA in these plants is about 20 times more abundant than in transgenic P nos-nptII plants.