Expression Dynamics Of Pheromone Binding Proteins And Expression Influences By Mating And Knockdown Of Cryl In Spodoptera Exigua

With the long time of evolution, insect evolves the complicated and highly effective chemosensory mechanisms, by which insect percepts the array of environmental chemical cues and subsequently respond with various physiological and behavioral responses, such as finding partner, food and habitats, and escaping from prayers. Sex pheromone communication system particularly in moths is extremely sensitive and species-specific, thus is thought as a perspective alternative to be exploited in pest control. Pheromone binding protein (PBP) takes crucial roles in chemical communication betwee males and females. In order to explore highly effective chemicals that interfere or block the communication between male and female, it is necessary to understand the mechanisms of PBP in sex pheromone perception. Based on the previous identification of three SexiPBPs, in the present study we further determined their expression dynamics, effect of mating on the PBP expression, and the possible regulation of PBP expression by circadian clock gene in spodoptera exigua. The main results were as follows:1. Temporal pattern and sex diffrences of PBP expression in S. exiguaThe temporal pattern and sex difference of PBP expression were determined by using qRT-PCR. In one photoperiod, expressions of PBP1 and PBP2 showed similar temporal patterns, increasing from mid-late scotophase, reaching the peak at the end of scotophase, and decreasing at photophase. However, PBP3 showed no obvious fluctuation in expression throughout the whole photoperiod. As for moths of different days after eclosion, three PBPs displayed similar expression patterns, with a lower level at - 2d, then gradual increase to 3d, a slight reduction at 4d, and afterwards with no changes.Three PBPs showed the different expression patterns between males and females. PBP1 expressed significantly higher in males than females (male/female≈7), whileas PBP2 and PBP3 was significantly higher in females than males. By comparing the expression levels among PBPs, it was found that expression of PBP 1 in males was about 10 times that of PBP2 and PBP3, but in females no significant difference was found among three PBPs. 2. Effect of mating on PBP and OR2 expression in S. exiguaMating is the terminate purpose of sex pheromone communication between male and female. To explore the mechanisms of mating effect on sex pheromone communication, effects of mating on the expressions of PBPs and OR2 were measured by qRT-PCR. The results showed that mating had no significant effect on expression of three PBPs and OR2 gene regardless of males and females, indicting that the mating experience has no effect on the perception ability of males to the female sex pheromones. This result is consistent to the multiple mating habits of male S. exigua.3. Molecular cloning and circadian expression patterns of clock genes in S. exiguaClock genes play important roles in organisms in adapting to the environments and maintaining the normal life activities. By using bioinformatics methods, some cDNA fragments of clock genes were obtained from the transcriptome data. The full-lengh cDNAs of six genes were further obtained by RACE techniques, and designated as SexiPer, SexiTim, SexiClk, SexiDbt, SexiCry1 and SexiCry2, with GenBank Accession No. of HQ731034, HQ731035, HQ731032, HQ731035, HQ234484 and HQ234485, respectively. Phylogenetic analysis showed that these clock genes shared high identities in amino acid sequence with homologous genes reported in other insects.In addition, the diurnal expression patterns of these six genes were invesitgated by qRT-PCR. Per and Tim genes shared similar patterns, with the expression increasing from late photophase and reaching the peak at the end of photophase, and maitaining high level in scotophase. The Cry1 and Cry2 also showed similar circadian pattern in their expression, but the relative expression level between these two genes were different. In the photophase, the Cryl expressed in higher level than that Cry2; whileas in soctophse the contrast. Clk gene showed a high expression in photophase and low in scotophase. In comparison with above five genes, Dbt presented slightest fluctuation in expression throughout the whole photoperiod, showing a W shape of temporal pattern to some extent.4. Effect of Cry2 knockdown on the expression of PBP1 in S. exiguaTo explore the possible regulation of clock genes on the expression of PBPs, RNA interference (RNAi) experiments were carried out, by injecting the Cry2 dsRNA in the male moths of 3d old. qRT-PCR detection at 48h after the injiection showed that expresson level of Cry2 was significantly inhibited (about 2Q%), while the expresson of Cryl increased significantly. This result indicated that the expression of Cryl was inhibited by that of Cry2. The expression of PBP1 was also significantly reduced in dsCry2 injected moths than that in dsGFP injected moths, but not significantly different from those of DEPC injection and no-injection control moths.