Construction Of A CDNA Library Of The Antenna Of Musca Domestica And Cloning Of Odorant Binding Protein CDNA

Odorant binding proteins are thought to play an important role in insect for perceiving odorant molecule. Housefly Musca domestica is a kind of important sanitation insect. The researches of olfaction principle and odorant binding protein supply latent value for sustainable management of housefly, which not only make us know the mystery of odorant world, but also bring crucial significance in ecology and economy. Our research provides a molecular biology flat roof and foundation for the research of olfaction principle of housefly: cloning of odorant binding protein cDNA fragments using RT-PCR (Reverse Transcription PCR) and construction of a cDNA library using the Creator? SMART? cDNA library construction kit (Clontech).Two cDNA fragments of two different odorant binding protein genes in Musca domestica are amplified with two pairs of specific primers by RT-PCR, and the lengths of them were 381bp named MdomOBP1(GenBank Accession:AY730350) and 353bp named MdomOBP2(GenBank Accession: AY730351) respectively. Sequencing and analysis show that these two odorant binding protein cDNA fragments are characterized by typical conservative Cys: CysI= CysIII, CysII= CysV, CysIV= CysVI. Deduced ammo acid sequences are highly similar to that of six odorant binding protein from Diptera, with sequence identities of 57%~88% for MdomOBP1 and 52%~91% for MdomOBP2. The result shows that the two cDNA fragments of two different odorant binding protein genes may be partial sequences coding odorant binding protein of housefly.In order to understand the molecular mechanism of olfaction of housefly Musca domestica, we construct a cDNA library using the Creator? SMART? cDNA library construction kit(Clontech).Total RNA is isolated from the housefly antenna using TRIzol Reagent. The PowerScript? reverse transcriptase is used to synthesize and anchor the first-strand cDNA. Following reverse transcription, LD-PCR is 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 are continued togenerate the oriental, unamplified cDNA library. The unamplified cDNA library has a titer of about 6.3xl08cfu/ml, in which the recombinant clones with an average insert size of 1.7 kb are 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. According to the quality criterion of a good cDNA library (Clontech), the cDNA library constructed in the experiment is excellent. At the same time, two pairs of primer I and pirmer II are used to amplify the cDNA library and the result coheres with the RT-PCR reaction.A full-length odorant binding protein cDNA with 5' and 3' untranslated regions is isolated from the cDNA library, and the length of it is 618bp named MdomOBP3(GenBank Accession:AY826189). Sequencing and analysis show that the OBP cDNA is characterized by typical conservative Cys: Cysl= CysIII, CysII= CysV, CysIV= CysVI. Deduced amino acid sequence is highly similar to that of six OBPs from Diptera, with sequence identity of 59%~82% .