|Mccombs, Susan - UNIV. OF HI, HONOLULU, HI|
Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 10, 2000
Publication Date: December 1, 2000
Citation: Handler, A.M., Mccombs, S.D. 2000. The piggyBac transposon mediates germ-line transformation in the oriental fruit fly and closely related elements exist in its genome. Insect Molecular Biology. 9:605-612. Interpretive Summary: The ability to achieve gene transfer in economically important insects is important to improve the efficiency of the sterile male release program for fruit flies. Development of this methodology depends on the analysis of efficient gene transfer vector systems, and an evaluation of their stability necessary for applied use. Previously, the piggyBac vector from the cabbage looper moth, was found to mediate germline transformation in the Mediterranean and Caribbean fruit flies. In order to determine if this system can function as well in other tephritid fruit fly pests, piggyBac genetransfer was tested in the Oriental fruit fly, Bactrocera dorsalis. Transformation was achieved efficiently, but control experiments revealed the unexpected finding that the piggyBac transposon also existed in the host species. This was verified for several Oriental fruit fly strains by the isolation and molecualr analysis of several piggyBac elements. This is the first discovery of piggyBac in a species other than the cabbage looper and it has important implication for the further study and potential application of insects transformed with the piggyBac vector.
Technical Abstract: Germ-line transformation of a white eye strain of the oriental fruit fly, Bactrocera dorsalis, was achieved with the piggyBac vector, derived from a transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The vector was marked with the medfly white gene cDNA, and three transgenic lines were identified at a frequency of approximately 2% per fertile Go. Vector integrations were verified by Southern DNA hybridization, which also revealed the presence of endogenous genomic elements closely related to piggyBac. Appoximately 10 to 20 elements per genome were evident in several B. dorsalis strains, and sequence analysis of 1.5 kb PCR products from two wild strains and the white eye host strain indicated 95% nucleotide and 92% amino acid sequence identity among resident elements and the T. ni element. PiggyBac was not evident by hybridization in other tephritid species, or insects previously transformed with the transpsoson. This is the first discovery of piggyBac beyond T. ni, and its existence in a distantly related species has important implications for the practical use of the vector and insects transformed with it.