|Handler, Alfred - Al|
Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/29/2009
Publication Date: 6/10/2009
Citation: Zimowaska, G.J., Nirmala, X., Handler, A.M. 2009. The ß 2-tubulin gene from three tephritid fruit fly species and use of its promoter for sperm marking. Insect Biochemistry and Molecular Biology. 30:508-515. Interpretive Summary: The ability to create transgenic strains of economically and medically important insect species has the potential to greatly enhance our ability to improve existing biological control methods and develop more novel means of control. In particular the existing sterile insect technique (SIT) could be improved by facilitating genetic sexing, male sterility, and sperm marking. A visible fluorescent marker expressed in sperm can be used for larval male selection, identification of mated females in the field, and for sperm precedence studies. Lethal gene expression could confer male sterility that would provide a major advance over radiation-induced sterility. A prime candidate to mediate such effects, is the B2-tubulin gene. Scientist at the USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology Gainesville, Florida, isolated the B2-tubulin gene from three tephritid fruit fly species, the Caribbean fruit fly, Anastrepha suspensa, the Mexican fruit fly, A. ludens, and the oriental fruit fly, Bactrocera dorsalis. In addition we isolated the closely related B1-tubulin gene, allowing structural and developmental comparisons.
Technical Abstract: To isolate testis-specific regulatory DNA that could be used in genetically transformed insect pest species to improve their biological control, B2-tubulin genes and their proximal genomic DNA were isolated from three economically important tephritid pest species, Anastrepha suspensa, A. ludens, and Bactrocera dorsalis. Gene isolation was first attempted by degenerate PCR on an A. suspensa adult male testes cDNA library, which fortuitously isolated the 2.85 kb B1-tubulin gene that encodes a 447 amino acid polypeptide. Subsequent PCR using 5’ and 3’RACE isolated the 1.4 kb AsB2-tubulin gene that encodes a 446 amino acid polypeptide. Using primers to conserved sequences, the highly similar A. ludens and B. dorsalis B2-tubulin genes, having identical amino acid sequences, were then isolated. To functionally identify the AsB2-tubulin gene, qRT-PCR showed that AsB2-tubulin transcript was most abundant in pupal and adult males, and specific to the testes. This was further tested in transformants having the DsRed.T3 reporter gene regulated by the AsB2-tubulin 1.3 kb promoter region. The fluorescent protein was specifically expressed in testes from third instar larvae to adults, and fluorescent sperm could be detected in the spermathecae of non-transgenic females mated to transgenic males.