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Leaf-feeding larvae of Manduca sexta (Insecta, Lepidoptera) drastically reduce copy numbers of aadA antibiotic resistance genes from transplastomic tobacco but maintain intact aadA genes in their feces

Published online by Cambridge University Press:  26 October 2007

Nicole Brinkmann  and
Christoph C. Tebbe
Nicole Brinkmann
Affiliation:
Institute of Agroecology, Federal Agricultural Research Centre (FAL), Bundesallee 50, 38116 Braunschweig, Germany
Christoph C. Tebbe
Affiliation:
Institute of Agroecology, Federal Agricultural Research Centre (FAL), Bundesallee 50, 38116 Braunschweig, Germany

Abstract

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The objective of this study was to evaluate the effect of insect larval feeding on the fate and genetic transformability of recombinant DNA from a transplastomic plant. Leaves of tobacco plants with an aadA antibiotic resistance gene inserted into their chloroplast genome were incubated with larvae of the tobacco hornworm Manduca sexta (Lepidoptera). The specifically designed Acinetobacter strain BD413 pBAB2 was chosen to analyze the functional integrity of the aadA transgene for natural transformation after gut passages. No gene transfer was detected after simultaneous feeding of leaves and the Acinetobacter BD413 pBAB2 as a recipient, even though 15% of ingested Acinetobacter BD413 cells could be recovered as viable cells from feces 6 h after feeding. Results with real-time PCR indicated that an average of 98.2 to 99.99% of the aadA gene was degraded during the gut passage, but the range in the number of aadA genes in feces of larvae fed with transplastomic leaves was enormous, varying from 5 × 106 to 1 × 109 copies.g-1. DNA extracted from feces of larvae fed with transplastomic leaves was still able to transform externally added competent Acinetobacter BD413 pBAB2in vitro. Transformation frequencies with concentrated feces DNA were in the same range as those found with leaves (10-4–10-6 transformants per recipient) or purified plasmid DNA (10-3–10-7). The presence of functionally intact DNA was also qualitatively observed after incubation of 30 mg freshly shed feces directly with competent Acinetobacter BD413 pBAB2, demonstrating that aadA genes in feces have a potential to undergo further horizontal gene transfer under environmental conditions.

Keywords

transplastomic plantsaadA genenatural transformationAcinetobacterinsect larvae
Type
Research Article
Information
Environmental Biosafety Research , Volume 6 , Issue 1-2: Thematic Issue on Horizontal Gene Transfer , January 2007 , pp. 121 - 133
Copyright
© ISBR, EDP Sciences, 2007

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