Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco.
A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5'-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.
Cardon
G H
GH
Max-Planck-Institut für Züchtungsforschung, Abteilung Molekulare Pflanzengenetik, Köln, Germany.
Frey
M
M
Saedler
H
H
Gierl
A
A
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA; D004251:DNA Transposable Elements; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics*; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*; D003313:Zea mays / Q000235:genetics
D004251:DNA Transposable Elements; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase