The β2-tubulin gene from three tephritid fruit fly species and use of its promoter for sperm marking

To isolate testis-specific regulatory DNA that could be used in genetically transformed insect pest species to improve their biological control, β2-tubulin genes and their proximal genomic DNA were isolated from three economically important tephritid pest species, Anastrepha suspensa, Anastrepha 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 β1-tubulin gene that encodes a 447 amino acid polypeptide. Subsequent PCR using 5′ and 3′ RACE generated the 1.4 kb Asβ2-tubulin gene that encodes a 446 amino acid polypeptide. Using primers to conserved sequences, the highly similar A. ludens and B. dorsalis β2-tubulin genes, encoding identical amino acid sequences, were then isolated. To verify Asβ2-tubulin gene identification based on gene expression, qRT-PCR showed that Asβ2-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 Asβ2-tubulin 1.3 kb promoter region. 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.To confirm these matings, a PCR protocol was developed specific to the transgenic sperm DNA.