DEFINITION AND CHARACTERIZATION OF AN ARTIFICIAL EN-BASED SPM-BASED TRANSPOSON TAGGING SYSTEM IN TRANSGENIC TOBACCO

Citation
Gh. Cardon et al., DEFINITION AND CHARACTERIZATION OF AN ARTIFICIAL EN-BASED SPM-BASED TRANSPOSON TAGGING SYSTEM IN TRANSGENIC TOBACCO, Plant molecular biology, 23(1), 1993, pp. 157-178
Citations number
71
Categorie Soggetti
Plant Sciences",Biology
Journal title
ISSN journal
01674412
Volume
23
Issue
1
Year of publication
1993
Pages
157 - 178
Database
ISI
SICI code
0167-4412(1993)23:1<157:DACOAA>2.0.ZU;2-R
Abstract
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 exc ision are expressed by fusing their cDNAs to the CaMV 35S promoter. Th e dSpm receptor component is inserted in the 5'-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed g ermination on L-PPT-containing medium or by spraying seedlings with th e herbicide Basta. Using this bar-based excision reporter construct, a n average frequency of germinal excision of 10.1% was estimated for dS pm-S, an En/Spm native internal deletion derivative. Insertion of En-f oreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSp m-DHFR has a lower frequency of somatic and germinal excision than dSp m-S. Revertants carrying a transposed dSpm-DHFR element can be selecte d with methotrexate. Germinal excision is frequently associated with r einsertion but, as in maize, dSpm has a tendency to integrate at chrom osomal locations linked to the donor site. Concerning the timing of ex cision, independent germinal transpositions are often found within a s ingle seed capsule. All activity parameters analysed suggest that tran sposon tagging with this system in heterologous hosts should be feasib le.