Successful gene tagging in lettuce using the Tnt1 retrotransposon from tobacco.
The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3beta-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.
Mazier
Marianne
M
Unité de Génétique et d'Amélioration des Fruits et Légumes, UR1502, Institut National de la Recherche Agronomique, F-84143 Montfavet cedex, France. mazier@avignon.inra.fr
Botton
Emmanuel
E
Flamain
Fabrice
F
Bouchet
Jean-Paul
JP
Courtial
Béatrice
B
Chupeau
Marie-Christine
MC
Chupeau
Yves
Y
Maisonneuve
Brigitte
B
Lucas
Hélène
H
D018745:Genome, Plant; D005966:Glucuronidase / Q000032:analysis; D018545:Lactuca / Q000033:anatomy & histology / Q000201:enzymology / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000033:anatomy & histology / Q000378:metabolism; D018626:Retroelements; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
D010940:Plant Proteins; D018626:Retroelements; D006899:Mixed Function Oxygenases; D005966:Glucuronidase