Immunolocalization Of Lymantria Dispar Pheromone Binding Proteins And Their Key Sites Residues Involved In Interacting With Chemicals

The gypsy moth, Lymantria dispar L., is a very dangerous crop and forest pest,which spr-ead widely in the world. Asian gypsy moth has been international quarantine pest because fem-ale moth could fly, host plants are wider, and has a great impact on international trade. The o-lfaction of insect plays an important role in its behavior. To date, the gypsy moth olfactory tra-nsmission mechanism is not fully clear. In order to research better the monitor and control tec-hnique of gypsy moth, and detect the functions of Lymantria dispar Pheromone binding protein-s(PBPs), the ultrastructure of the antennal chemosensilla were observed on scanning and transm-ission electron microscopy; the immunolocalization of LdisPBP1and LdisPBP2in chemosensilla on antenna were characterizated by immunocytochemitry; the possible sites residues involved in interacting with chemicals of LdisPBPs were predicted by bioinformatics technology; based on the predicted results, we designed mutants of PBP1and PBP2by site-directed mutagenesis and detected key sites residues involved in interacting with chemicals by competitive fluorescence binding assays. The main results are as followings:(1)Ultrastructure of antennal chemosensilla in Lymantria disparThere are four types chemosensilla in both male and female mothes antennae, including se-nsilla trichodea, sensilla basiconica, sensilla coeloconica and sensilla chaetica. Sensilla trichodea have thick single-wall.There are relatively few pores on the wall. Sensilla basiconica have thin single cuticular wall with numerous wall pores. Sensilla coeloconica with edgehair are situated in pits on the antennal surface. It has longitudinal ridges on the surface and double sensillar walls perforated by radial pores. Sensilla chaetica have a thick non-porous wall and a terminal pore.(2)Immunolocalization of PBPs from Lymantria dispar in antennal chemosensillaImmunocytochemical experiments revealed that anti-PBP1and anti-PBP2are specifically la-baelde in the sensillum lymph of sensilla trichodea in male antennae and both sensilla basiconi-ca and sensilla trichodea in female antennae.(3)Possible sites residues involved in interacting with chemicals of LdisPBPsAll the residues of LdisPBPl and LdisPBP2models were mutated to alanine using bioinfo-rmatics techmology, and the mutation energy of every amino acid residues were calculated. The top three residues which cause to effect of destabilizing were selected to be possible sites res-idues involved in interacting with chemicals of LdisPBPs. The residues of LdisPBPs models int-eracing with chemical ligands in docking simulations were also selected to be possible sites re- sidues. The results showed that to PBP1, phenylalanine at position36and lysine at position94could be possible sites residues interacting with chemicals. To PBP2, glycine at position49, phenylalanine at position76could be the possible sites residues interacting with chemicals.(4)Key sites residues involved in interacting with chemicals of LdisPBPsWe designed4LdisPBP1mutants F36A、Y41A、F76A. K94A and3LdisPBP2mutants G49A、F76A、K121A. The binding ability of mutants with chemicals was detected by compete-tive fluorescence binding assays. The results showed that to PBP1, phenylalanine at position36and lysine at position94could be key sites residues interacting with chemicals. To PBP2, gl-ycine at position49, phenylalanine at position76could be key sites residues interacting with chemicals.