RNAi Of Pheromone Binding Protein And Tissue Expression Survey Of Olfactory Receptor In Spodoptera Exigua Hübner

In insects, the olfactory systems have been evolved to be highly sensitive, enabling insects to detect and discriminate among a diverse array of volatile chemicals. With this sensitive olfactory system, insects resopond properly to the chemical environment by different behaviors, such as mate-seeking, food and habitat locating, and oviposition-site finding. Therefore, the olfactory-based control strategies have developed to be an important means for pest control. To furter improve the olfactory-based control strategies, it is very important to gain insight on the molecular mechanism of the perception of female sex pheromone by the male. With the insights on the function of olfactory related protein, it will be facilitated for molecular designing of specific regulator targeted to these olfactory proteins, and for further development of more effective pest behaviorally interference techniques.Pheromone binding proteins (PBPs) and olfactory receptor (Ors) are thought to play critical roles in male perception of conspecific female sex pheromone. The major method to address the functions of PBP is in vitro binding assays between PBP and sex pheromone chemicals. As a robust method to study the new protein or gene functions, RNA interference (RNAi) has been used in many organisms including plants and animals. However, there is no report to date on the PBP fuction study with the employment of the RNAi method. Based on our previous molecular characterization of the PBP and OR2 genes in Spodopera exigua and S. litura, the present paper dealt with the PBP function study by using RNA interference technique. In addition, the expression pattern of SexiOR2 and SlitOR2 genes among different insect tissures of S. exigua and S. litura were inversitated using RT-PCR method. The main results were as follows.1) Fragment of 530bp was amplified with the specific primers designed accoding to known cDNA sequence of Sexig PBP1, and the dsRNA of Sexig PBP1 used for RNAi was further synthesized with above 530bp fragment as template using T7 RiboMAX Express RNAi System. 2) Abodomen injection method was tested for introducing SexigPBP1 dsRNA into the moth abdomen cavity. RT-PCR and qRT-PCR detections at two days after injection of dsRNA revealed that the mRNA expression level of SexigPBP1 in male antenna was significantly reduced by about 90%, and that the moth survival rates were not significantly different between treatments of dsRNA injected and DEPC-water injected. Therefore, the abodomen injection method was effective to be used for PBP RNAi in the moth species.3) Two days after dsRNA injection, the electroantennography (EAG) mesurment was further performed to detect the response of live moth at electrophysiolocial level. The results showed that the EAG response to the major femal sex pheromone component Z9,E12,14:Ac was reduced significantly by about 60% in dsRNA injected moths comparied to the DEPC-water injected control moths, which provided the direct evidence that SexigPBP1 played important role in the sex pheromone perception in male moths. This is the first report on investigation of insect PBP function in vivo by using RNAi approach.4) The tissure expression patterns of SexiOR2 and SlitOR2 were investigated, respectively, in S. exigua and S. litura by RT-PCR approach. As a result, the antenna of both sexes was the unique tissur to express the OR2 gene, while the head, thorax, abdomen, wing, proboscis and leg had no detected expression of the OR2 gene.