Construction of a cDNA Library of the Antenna of Housefly, Musca domestica

In order to understand the molecular mechanism of olfaction of housefly, Musca domestica, we constructed a cDNA library using the CreatorTM SMARTTM cDNA library construction kit (Clontech). Total RNA was isolated from the housefly antenna using TRIzol Reagent. The PowerScriptTM reverse transcriptase was used to synthesize and anchor the first-strand cDNA. Following reverse transcription, LD-PCR was performed using a modified oligo (dT) and an anchor primer to enrich the cDNA population for full-length sequences. cDNA size fraction, ligation and transformation into E. coli were continued to generate the oriental, unamplified cDNA library. The unamplified cDNA library has a titer of about 6.3×108 cfu/mL, in which the recombinant clones with an average insert size of 1.7 kb were about 91%. Amplification of oriental library is vital to obtain enough high quality plasmid for library screening and for long-term storage of the library. A full-length odorant binding protein (OBP) cDNA with 5′ and 3′ untranslated regions was isolated from the cDNA library, and named MdomOBP3 (GenBank Accession: AY826189). Sequencing and analysis show that the OBP cDNA is characterized by typical conservative Cys. Deduced amino acid sequence is highly similar to that of six OBPs from Diptera, with sequence identity of 59%-82%.