Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

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Abstract:

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 ± 1.4 transformants per plate could be obtained using conidial concentrations of 106 conidia/mL and a 2 day co-cultivation in the presence of 200 µmol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.Key words: entomopathogenic fungi, Metarhizium anisopliae, Agrobacterium tumefaciens, genetic transformation.

Une méthode de transformation par l'intermédiaire de Agrobacterium tumefaciens simple, hautement efficace et fiable fut élaborée pour le champignon entomopathogène Metarhizium anisopliae. L'expression du gène de la protéine fluorescente verte, egfp, et du gène de la résistance au bénomyl, benA3, ont été employées comme marqueurs de M. anisopliae transformés. Les efficacités de transformation dépendaient de la souche de A. tumefaciens utilisée. Avec la souche AGL-1, 17,0 ± 1,4 transformants par plaques ont pu être obtenus en utilisant des concentrations de conidies de 106 conidie/mL est une co-culture de 2 jours en présence de 200 µmol/L d'acétosyringone. D'un autre côté, les transformations avec la souche LBA4404 ont échoué. Dix transformants furent analysés par hybridation de type Southern et ont révélé une seule copie d'ADN T. Vingt transformants ont été sous-cultivés pendant cinq générations sur du milieu non sélectif et 95 % des transformants étaient mitotiquement stables. La transformation de M. anisopliae par l'intermédiaire d'A. tumefaciens pourrait être utile à l'étude de gènes impliqués dans la pathogénicité chez l'insecte.Mots clés : champignons entomopathogènes, Metarhizium anisopliae, Agrobacterium tumefaciens, transformation génétique.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: July 1, 2006

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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