Transgenic Expression Of The Tsetse Fly Glossina Fuscipes Fuscipes Olfactory Receptor, Or67d, In Drosophila Melanogaster
Tsetse flies are the sole vectors of Human African trypanosomiasis (sleeping sickness) and Animal African trypanosomiasis (Nagana). The insect is attracted to its suitable hosts through external signals which are perceived by olfactory receptors (ORs); thus representing the basis of transmission of the disease to thousands of people and millions of livestock. A developing approach to efficiently identify the key chemical ligands of odorant receptors entails expressing single ORs in different cell systems for consequent screening analysis. This study aimed to establish the expression of an expanded olfactory receptor family, Or67d of Glossina fuscipes fuscipes, a vector of both animal and human trypanosomiasis, in a Drosophila system. The receptor homologue is known to mediate responses to Drosophila melanogaster male-specific pheromone 11-cis-vaccenyl acetate (cVA) regulating mating behavior of males and females. In G. f. fuscipes, five copies of the same gene were found to be homologous to Or67d of Drosophila melanogaster. Out of the five copies, four were typically complete and only three of them contained the conserved seven-transmembrane-helix 6 (7tm_6) odorant receptor domain. Phylogenetic reconstruction of the four gene copies suggested a closest relationship between GffOr67d4 and Drosophila homolog, DmelOr67d. This gene copy was synthesized in pUC57 vector, amplified by polymerase chain reaction, cloned in pENTRTM/D-TOPO® vector then sub-cloned into the destination vector pTW. Sequencing analysis using Bioedit v.184.108.40.206 revealed that the gene was successfully cloned between attB sites, downstream of the upstream activating sequence (UAS). Afterwards, the recombinant plasmid was injected in Drosophila embryos by fly genetic services. Transgenic flies presenting red eyes were subjected to RNA extraction, cDNA synthesis and RT-qPCR analysis. All RT-qPCR performed on the Drosophila transgenic flies both males and females showed that our gene of interest GffOr67d4 was expressed in Drosophila relative to the internal control, alpha-tubulin. Our study revealed that the Drosophila system can actually be used as a heterologous cell system for the identification of behavioral and ecologically relevant chemical signals of ORs in tsetse fly species and for the design of olfactory-based strategies to control trypanosomiasis.
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