Continuous transposition of the maize Ac element in four generations of transgenic tobacco

--Reinhard Hehl and Barbara Baker

We have studied the activity of the maize transposable element Activator (Ac) in transgenic tobacco plants. Six transposed Ac elements have been cloned and are found to be integrated into and close to unique and low copy DNA. Five of these elements have been cloned from an R1 plant obtained through selfing of an original R0 transformant. These elements are designated Nt-1::Ac-18, Nt-5::Ac, Nt-6::Ac, Nt-7::Ac and Nt-8::Ac. Four of the five elements isolated from the R1 plant (Nt-5::Ac, Nt-6::Ac, Nt-7::Ac and Nt-8::Ac) can not be detected in genomic Southern blot hybridizations. Since the R1 plant does not contain Ac sequences in T-DNA this suggests that transposed elements continue to transpose somatically. One element (Nt-1::Ac-18) is stable and can be detected in genomic Southern blots in the R1 as well as the R0 plant. Ac-18 sustained a 4bp terminal deletion and has never been found to transpose again.

The induction of an 8bp duplication of target sequences of Ac (Nt-6::Ac) upon integration reveals that the mechanism of integration of Ac in tobacco is similar to that in maize. The result that the R1 plant harbored transposing Ac elements has been verified by analyzing R2 offspring plants obtained through selfing of the R1 plant. Genomic Southern blot hybridizations showed that 10 out of 12 R2 plants harbored new and different Ac homologous restriction fragments distinct from those present in the R1 progenitor. This indicates that Ac has transposed frequently into new and unique target sites. To confirm that the new restriction fragments represent newly transposed Ac elements we cloned one of these elements (Nt-2::Ac) from an R2 plant harboring only Nt-1::Ac-18 (see above) and Nt-2::Ac. The presence of an 8bp duplication adjacent to Ac at Nt-2::Ac reveals that Ac indeed transposed into this particular target fragment. Analysis of R3 offspring plants isolated by selfing of the R2 plant harboring only Nt-2::Ac and the stable Nt-1::Ac-18 demonstrates that the Ac element at Nt-2::Ac is capable to transpose again, since new Ac homologous restriction fragments can be detected in genomic Southern blot hybridizations. Molecular analysis of tobacco DNR adjacent to Ac in Nt-2::Ac shows the presence of not only unique but also repetitive DNA sequences. Furthermore a transcript homologous to the unique DNA fragment of Nt-2::Ac can be detected in Northern blot hybridizations.

The observed continuous activity of Ac in transgenic tobacco also correlates with the detection of the Ac specific 3.5kb mRNA as well as with the lack of methylation of Ac internal sequences. The latter was analyzed by HpaII and PvuII digestion of DNR of the RO plant as well as 4 R1 and 12 R2 plants obtained through selfing of the RO plant. All 17 plants harbor Ac elements at different target sites and in none of these plants were Ac sequences found to be methylated.

These data suggest that Ac can be employed for transposon tagging in transgenic tobacco. We are currently using Ac in an attempt to tag the dominant TMV resistance gene N.

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