En/Spm as a tool for gene tagging in heterologous species

--Guillermo H. Cardon, Monika Frey, Heinz Saedler and Alfons Gierl

An excision assay system for En/Spm in transgenic tobacco defined recently (Frey et al., EMBO J. 9:4037-4044) helped to identify the transposon-encoded trans-acting functions (TNPA and TNPD) necessary for excision. In this system, excision and reinsertion of a 2.2kb dSpm element, a natural internal deletion mutant of the autonomous element, takes place with similar characteristics as En/Spm transposition in maize.

This system was modified in order to tailor it for gene tagging. A 4.2kb receptor element marked by the insertion of a DHFR gene, which confers resistance to methotrexate, was constructed. This artificial element is capable of normal excision and reinsertion in dependence of TNPA and TNPD in transgenic tobacco. New excision reporter constructs were made in which the elements are inserted in the untranslated leaders of the selectable marker genes bar and Spt. The timing and frequency of excision can be manipulated by expressing the trans-acting factors under the control of different promoters. The bar gene proved to be an excellent marker for the selection of germinal revertants among tobacco seeds germinating in vitro in the presence of 100mg/l L-PPT. Selection of germinals is also possible in the greenhouse by spraying seedlings with the herbicide BASTA (Hoechst AG). When TNPA and TNPD were expressed from CMV 35S promoter, the average frequency of germinal excision observed for the 2.2kb dSpm from a bar reporter was about 25%. In some plants, an early excision of the dSpm resulted in 100% of the gametes carrying the same reversion event. The marked receptor element has both lower somatic and germinal excision frequencies but this disadvantage is compensated for by the fact that it can be selected for reintegration.

Tobacco proved to be a suitable test system to study and manipulate En/Spm transposition, but since it is amphidiploid it is not a convenient species for transposon tagging. Two diploid species were chosen, Petunia and Arabidopsis, in which a working transposon tagging system for gene isolation would be desirable. Excision reporters and trans-activating constructs previously tested in tobacco were transformed into Petunia and Arabidopsis and are presently being joined by sexual crossing. These two species were also transformed with the autonomous element En-1. It was found to transpose in Arabidopsis in all the independent transformants analyzed as well as in the two subsequent generations. The frequency of somatic excision is over 50% and footprints left behind are normal. The autonomous element is capable of activating the 2.2kb dSpm with high frequency. Preliminary experiments show that the frequency of germinal excision of En-1 in Arabidopsis is about 3%. Concerning En-1 activity in transgenic Petunia, none of the four independent transformants analyzed so far showed excision.

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