Identification And Fuctional Analysis Of The Odorant Binding Protein Genes From Plutella Xylostella

The diamondback moth(DBM),Plutella xylostella(L.)(Lepidoptera:Plutellidae)is a notorious insect pest that attacks cruciferous plants.DBM is widely distributed in China and in southern provinces its damage is particularly serious.With the increase in large-scale cultivation of cruciferous vegetables in the north,DBM is causing large economic losses due to the rapid development of pesticide resistance.Olfaction plays an important role in insect physiology and behavior.Insects perceive an environment that is full of complex chemical pheromones through their olfactory system;a system that affects the insect's mating,feeding,habitat seeking,and locating of plants hosts functions.Odorant binding proteins(OBPs)are involved in the first step of identifying environmental and biochemical reactions.Elucidating the function of OBPs is fundamental to our understanding of insect olfactory mechanism.This study focused on identifying 5 OBPs genes of DBM and their functions through cloning,prokaryotic expression,fluorescent competitive binding and homology modeling.The results of our study are outlined below:1)We cloned five OBPs genes using PCR,and named them PxylGOBP1,PxylGOBP2,PxylOBP1,PxylOBP2 and PxylOBP3(GenBank ID:KT070561?KT070564,KT070560,KT070562 and KT070563,respectively),The cDNA lengths of the genes are 501,492,345,546,465 bp,and code for 167,164,115,182,and 155 amino acids,respectively.The predicted molecular masses are 19.53,18.57,12.73,20.98 and 17.47 KDa,with isoelectric points of 5.65,5.12,9.10,5.23 and 9,respectively.Signal peptide analysis the N-terminal shows that PxylGOBP1,PxylGOBP2,and PxylOBP3 contain 22,22,and 16 amino acids,respectively,but PxylOBP1 and PxylOBP2 have none.Blast of the 5 sequences shows that all of the OBPs possess six conserved cysteines(C1-X23-30-C2-X41-42-C4-X10-13-C5-X8-C6)and,thus,are "Classic" OBPs.We constructed a neighbor-joining using MEGA5.1 software and found that the similarity between PxylOBP1,Spodoptera exigua SexiOBP20 and Spodoptera litura SlitOBP19 is the highest,followed by that of PxylGOBP1 and Amyelois transitella AtraGOBP1.The similarity between PxylOBP2 and Bombyx mori BmorOBP2 is the highest,followed by that of PxylGOBP2 and Arguresthia conjugella AconGOBP2;PxylOBP3,Spodoptera litura OBP32 and Operophtera brumata OBPa,were the closest.2)We constructed the prokaryotic expression vector pET28a-PxylOBPs and successfully expressed it in BL21(DE3).We obtained recombinant proteins through optimization of IPTG induction conditions.We used a nickel column purification system to obtain purified proteins.We used western blot analysis to confirm that the expressed products are the target proteins.A circular dichroism spectrum of the proteins demonstrated that the secondary structure is mainly alpha helices.3)We measured the binding efficiency of PxylOBPs with 39 different odorants using fluorescence competitive binding tests.The five OBPs exhibited different binding capacities to different odorant ligands.PxylGOBP1 has a strong binding ability with pheromone,indicating that it plays an important role in mate selection.PxylGOBP2 binds to most of the tested odorants,implying that it plays an important role in detecting both sex pheromones and plant volatiles' PxylOBP1 also bound(Z)-11-hexadecenal,Hexyl alcohol,Capryl alcohol,p-Ocimene and Geranyl acetate but could not bind two other sex pheromones,indicating that PxylOBP1 may play a role in locating host plants.Additionally,PxylOBP2 also bound(Z)-11-hexadecenal and 11 plant volatiles and has a strong binding capability towards Linalool and 1-Nonanol,implying that it plays an important role in the insect's courtship process.PxylOBP3 could bind the 3 sex pheromones and most of the aldehydes,indicating that PxylOBP3 might function in perception of both host and repellant volatiles.4)We constructed the three-dimensional structure of the PxylGOBPs using homology modeling and used it to predict the binding sites,Our results show more hydrophobic binding pocket in PxylGOBP2 than in PxylGOBP1 indicating a broader binding spectrum.We analyzed binding sites using the I-TASSER server and found that Ile52,Ser56,His70,Thr73,Met90,Glu98,Ile111 and Ile114 are the binding sites for PxylGOBP1,and that Thr9,Ile52,Ser56,Met62,Met73,Met90,Glu98,Val111 and Thrl 14 are the binding site for PxylGOBP2.