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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICALLY-BASED TECHNOLOGIES FOR MANAGEMENT OF CROP INSECT PESTS IN LOCAL AND AREA-WIDE PROGRAMS

Location: Insect Behavior and Biocontrol Research Unit

Title: Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements

Authors
item Raphael, K -
item Shearman, D -
item Streamer, K -
item Morrow, J -
item Handler, Alfred
item Frommer, M -

Submitted to: Genetica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 6, 2010
Publication Date: January 1, 2011
Citation: Raphael, K.A., Streamer, K., Morrow, J.L., Handler, A.M., Frommer, M., Shearman, D.C. 2011. Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements. Genetica. 139:91-97.

Interpretive Summary: The ability to achieve gene transfer in economically important insects, tephritid species in particular, for development of improved stran for the sterile insect technique, is a major goal of our laboratory at the CMAVE.Development of this methodology depends upon the discovery and analysis of efficient and stable gene transfer vector systems.Previously, the piggyBac vector from Trichoplusia ni was found to mediate germline transformation in the Mediterranean fruit fly, Ceratitis capitata.In order to determine if this system can function as well in other tephritid fruit fly pests, piggyBac gene-transfer was tested in the Queensland fruit fly, Bactrocera tryoni.To optimize the frequency of gene-transfer as well as the selection of transgenic insects, a highly expressed heat-shock regulated transposase helper gene was used with DsRed and EGFP fluorescent protein marker genes for transformant selection and detection in the field.A transformation frequency of approximately 5–10% was achieved and transgene inheritance remained stable for at least eight generations.Endogenous piggyBac-related sequences were observed in the B. tryoni genome, similar to those reported for the B. dorsalis complex of fruit flies and two noctuid moths.

Technical Abstract: We report the stable genetic transformation of the Queensland fruit fly Bactrocera tryoni using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP.A transformation frequency of 5–10% was obtained.Inheritance of the transgenes has remained stable over eight generations despite the presence of endogenous piggyBac sequences in the B. tryoni genome.The sequence of insertion sites shows the usual pattern of piggyBac integraton into TTAA target sites.An investigation of endogenous piggyBac elements in the B. tryoni genome reveals the presence of sequences almost identical to those reported recently for the B. dorsalis complex of fruit flies and two noctuid moths, suggesting a common origin of piggyBac sequences in these species.The availability of transformation protocols for B. tryoni has the potential to deliver improvements in the performance of the Sterile Insect Technique for this pest species.

Last Modified: 9/1/2014
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