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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Insect Behavior and Biocontrol Research » Research » Research Project #437806

Research Project: Creation of New Embryonic Transactivator Drivers for the Mediterranean Tephritid Fruit Fly

Location: Insect Behavior and Biocontrol Research

Project Number: 6036-22000-034-062-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Mar 1, 2020
End Date: Feb 28, 2022

The objective of this cooperative research project is the development of transgenic strains of the Mediterranean fruit fly (Medfly), Ceratitis capitata, for improved biological control of this important insect pest of fruit crops. This will be achieved by incorporating a tetracycline-suppressible lethality system, that acts specifically during embryonic development, into the genome of this pest species. A critical component of this system is the use of gene promoters, necessary for expression of a lethal effector, that function specifically during post-zygotic embryogenesis. This will be achieved by identifying and isolating the nullo and CG14426 gene promoters and linking them to the tetracycline-transactivator (tTA) within a piggyBac transposon vector, and transforming the vector into the host species.

Cognates to the early embryonic nullo and CG14426 genes will be identified by orthologous sequences to known nullo/CG14426 alleles in related species and isolated by direct internal polymerase chain reaction(PCR) and inverse PCR to genomic DNA from the Medfly. Upstream sequences containing putative regulatory promoter sequences will be isolated and linked to the tet-transactivator (tTA), with the complete nullo/CG14426-tTA cassettes inserted into a piggyBac transposon vector. The piggyBac vectors will then be transformed into a Medfly host strain, with G1 transformant insects selected by a fluorescent protein visible marker that will then be backcrossed to the parental lines. Fluorescent-marked offspring from each G1 line will then be inbred to create homozygous lines that will then be crossed to homozygous Tetracycline Response Element(TRE)-hid lethal effector lines with progeny tested for viability on diet with and without tetracycline.