Characterization of the proteasomeβ2 subunit gene and its mutant allele in the tephritid fruit fly pest, Anastrepha suspensa

In Drosophila melanogaster the beta 2 proteasome subunit gene, Pros beta 2, was first identified as a dominant temperature sensitive mutant, DTS-7, that causes pupal lethality at 29 degrees C but allows survival to adulthood at 25 degrees C. To explore the use of proteasome mutations for a conditional lethal system in insect pests, we identified and isolated the beta 2 subunit gene of the 20S proteasome from the Caribbean fruit fly, Anastrepha suspensa. The caribfly ortholog AsPros beta 2 was isolated from pupal cDNA by 5' and 3' RACE. The AsPros beta 2 protein has high amino acid sequence similarity to predicted insect Pros beta 2 subunits and homologs from yeast and mammals, and it contains the well conserved amino acids that confer catalytic activity and substrate specificity. AsPros beta 2 is a single copy gene and its RNA accumulates throughout all developmental stages of the caribfly. For functional studies a point mutation, analogous to the Pros beta 2(1) mutation in D. melanogaster, was introduced into AsPros beta 2 to create an aberrant protein with a Gly170Arg substitution. Consistent with the DTS-7 mutation, transgenic insects carrying the mutant allele undergo normal metamorphosis at the permissive temperature (25 degrees C) but at the non-permissive temperature (29 degrees C) they exhibit effective pupal lethality. This is the first report of a functional characterization of a Pros beta 2 cognate based on the creation of a dominant temperature-sensitive mutation. This type of temperature-dependent lethality could be used for biological control, where transgenic insects are reared to adulthood at 25 degrees C or lower and then released into the field where ambient temperatures averaging 29 degrees C or greater cause lethality in their progeny.