| In order to locate and evaluate food, shelter, mates, and breeding substrates as well as to avoid predators and other dangers-insects rely on an acute olfactory system. The rice leaffolder, Cnaphalocrocis medinalis (Guenee) is an important migratory pest to rice. Because of the larvae damage plants by folding the leaves and scraping the green leaf tissues within the fold, chemical control costs a lot of money, and can easily cause pollution to the environment. Several studies had shown that the rice leaffolders’female moths release a blend of sex pheromones to attract male moths over long distances, and male moths detect the released pheromones with extreme sensitivity and selectivity, but at the moment on the molecular mechanism of olfactory reception about the rice leaffolder is still unclear. Here, we study the pheromone binding proteins (PBPs) of the rice leaffolder, on the basis of genes cloning, we analysis the PBP genes expression pattern and the binding properties with the odorant. The results could provid a theoretical basis on clear the molecular mechanism of olfactory reception in the rice leaffolder and the further developing effective attractant. The main results are as follows:1Molecular characterization and sequence analysis of PBPs from C. medinalisThrough the technique of RACE and RT-PCR, we obtained three full-length PBPs genes (CmedPBP2, GenBank:KC507181; CmedPBP3, GenBank:KC507184and CmedPBP4, GenBank:KC507185) from the C. medinalis antennal cDNA library. CmedPBP2cDNA contained a495bp open reading frame that encoded a protein of164amino acids with the first21-amino acids predicted to be a signal peptide. CmedPBP3cDNA contained a549bp open reading frame that encoded a protein of182amino acids with the first22-amino acids predicted to be a signal peptide. CmedPBP4cDNA contained a444bp open reading frame that encoded a protein of147amino acids with no signal peptide. The multiple sequences alignment of the CmedPBPs with other Lepidoptera PBPs (full-length sequences from GenBank) showed high sequence identity (46.13%), but the homology of these three CmedPBPs were very low, only22.76%, suggested they belong to different groups. 2Temporal and spatial expression profiles of CmedPBPs genesThe results of qRT-PCR showed that:the transcript levels of the CmedPBPs were all very low in the pupal stage. The expression levels of PBPs were high in the moth antenna but very low in the other tissues. The expression levels of PBPs were much higher in male moths antenna than in females, especially for the CmedPBP2(The expression level of CmedPBP2in0-day-old male antennae was about95-fold higher than in females). The expression levels of three PBPs in0-d-old and3-d-old male antenna were higher than in1-d-old (p<0.05). The expression levels of the three PBPs in female antenna were also affected by the age of moths, for the highest expression of CmedPBP2, CmedPBP3and CmedPBP4was observed in1-day-old,0-day-old and5-day-old respectively, while the mated status of the moths didn’t affect the expression levels of the three PBPs in female antenna.3The prokaryotic expression, purification and fluorescence competitive binding assays of CmedPBPsThe three genes were constructed into bacterial expression vector pGEX-6p-1, pET-28a(+) and pET-22b(+), but only pET-22b(+)-CmedPBP4successfully over expressed in E.coli BL21(DE3) plysS and mostly present as inclusion bodies. After isolation, solubilization and refolding, extracts were purified with a DE52affinity column.Fluorescence competitive binding assays were applied to explore the binding characters of CmedPBP4with the32kinds of rice volatiles, used1-NPN as a probe. CmedPBP4showed binding abilities with ten rice volatiles, moreover, all of them are terpenes. Combining ability from strong to weak was followed by:(-)-trans-Caryophyllene,(Z)-Famesene,(-)-a-Cedrene,(1R)-(+)-a-pinene, Lonone, β-Myrcene, Cedrol (KD<20), Nerolidol, y-Terpinene and R-(+)-Limonene (KD>20), suggesting a role of CmedPBP4in general odorant chemoreception. |
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