| Olfaction plays a crucial role in guiding insects’ behaviors.Antenna is one of the most important olfactory organ of insects,which can sense chemical signals.Just because of this,insects can guide their interactions with plants and insects same or different species,such as,host-seeking,mating,oviposition and avoiding predators and so on.Odorant binding proteins(OBPs)and odorant receptors(ORs)have key roles in these processes.The bark beetle Dendroctonus armandi Tsai and Li is one of the most important forest insects,which causes considerable economic damage to forest each year in China,especially,in the area of Qinling and Ba Mountains.D.armandi as the pioneer only attacks the healthy Pinus armandi over 30 years old.In order to know the mechanism of D.armandi’s host seeking,key genes of D.armandi involved in the processing of olfactory signals were studied in this dissertation,and the results were listed as follows:1.We cloned fifteen c DNA sequences of odorant-binding proteins(OBPs)from D.armandi,named Darm OBPl～15.Blast P showed that all of the eleven OBPs belonged to PBP-GOBP family;CDD prediction showed that 9 of them have the structural domain PBP-GOBP(which belongs to PBP-GOBP family),OBP2 also have the structural domain ph BP(which have role in binding insect pheromone);7 of the 11 OBPs were classified into Classic OBP(contain 6 conserve cystine),the rest blenged to Minus-C OBPs(contain 4conserve cystine)by using multiple alignment analysis.2.The phylogenetic relationship(Distance Tree and Neighbor-joining Tree)showed that15 OBPs were separated into two groups: Classic OBP and Minus-C.OBP1-4,OBP6,OBP8,OBP9 and OBP15 belonged to Classic OBP group and the others belonged to Minus-C group.3.The transcript levels of Darm OBP1-15 in different tissues(including antennae,heads(without antennae),thoraces,abdomens,legs,and wings)were determined using real-time PCR.Statistical significant differences were found between tissue distributions of adult D.armandi of both sexes.Darm OBP1-5,8,11,12 and 14 were expressed highly in the antennae in both sexes.While Darm OBP1-4 and 8 were expressed at low levels in legs and wings,and Darm OBP11,12,and 14 were expressed in low levels in the heads,abdomens and wings.Darm OBP5 was exclusively expressed in adult antennae.OBP6 was expressed in both abdomens and wings while OBP7 was significantly expressed in antennae and wings of both sexes.OBP13 was expressed exclusively in wings while OBP9,10 and 15 were expressed in all the tissues,but showed higher expression levels in abdomens,wings,and antennae.4.We compared the relative abundance of each OBP transcript between male and female adult D.armandi antennae.The most important result was that OBP2-4,5,8,9 and 12 had significantly higher expression in male than female antennae.By contrast,OBP7 and 11 had a significantly higher expression in females than in males.For the other OBPs,there was no significant difference of expression levels between males and females.5.The transcript levels of Darm OBP1-15 in different development stages(larvae,early pupae,later pupae,female adult,male adult)were determined using real-time PCR.Darm OBPs showed statistically significant differences in transcript expression levels among different development stages.All of the 15 OBPs,except OBP11,are expressed in adults of both sexes.Eight OBPs are expressed though the whole life(7,8,9,10,12,13,14 and 15),but one OBP(11)is undetected in adults.Several OBPs are expressed more highly in adult males and females compared to other life stages(1,2,3,4,5,6 and 13).Two of them(3 and 5)are detected only in adults.Seven OBPs are highly expressed in pupae(7,11 and 14 are expressed more higher in early pupae;8,9,10 and 15 are expressed higher in later pupae).6.We successfully constructed prokaryotic expression vectors of 11 OBPs(OBP1-8-p GEX-4t-1 and OBP13-15-p ET-32a)and the expression condition such as temperature,IPTG concentration,and induction time of different plasmids has been explored.The recombinant proteins were separated from the Bac Ready-Protein Extraction Solution and then further purified.At last we obtained four OBPs(OBP4,OBBP5,OBP6 and OBP7)which have very high purity.7.Homology modeling results showed that the three-dimensional structures of 9 proteins(except OBP6 and OBP15,the scores of SWISS-MODEL were not satisfactory)have six αhelix,and α1,α3,α4,α5 and α6 constituted the hydrophobic binding pockets.We chosed 86 volatiles in order to explore the common characteristic of the binding sites of the 10 OBPs,of which the 3D-modle have been made.Based on the results of docking characteristics of 6volatiles,we found that,the predicted key binding sites for Darm OBP1-5 are THR79,TYR105,HIS112,TYR113 and PHE114;TRP56,PHE103,PHE116,PHE117 and VAL118;TYR54,GLN75,TYR120,PHE121,LEU122 and PRO123;TYR105,ALA112,TYR113 and MET114;PHE26,PHE52,VAL80,VAL102 and PHE105,respectively.The key binding sites for Darm OBP7,8 13,14 are PHE55,TYR106 and HIS113;LEU54,GLY55,ARG118 and THR119;LEU59,GLU75 and ARG76;THR76,ASN84,PHE106 and LEU118,respectively.8.We knocked down the Darm OBP genes(except Darm OBP2,OBP13 for which the concentration of ds RNA was too low)by RNAi to further study their role in odor response.The transcript levels of Darm OBPs in the non-injected or water-injected D.armandi remained unchanged.There was no obvious difference between water-injected and ds RNA-injected groups 24 h or 48 h after ds RNA injection.But in 72 h treatments,the expression levels of Darm OBPs were reduced by ～80% compared with water-injected control.RNAi only reduced expression of the target OBP indicating that expressions of other OBPs were not impacted.We examined the responses of ds OBPs-injected,water-injected,and non-injected D.armandi(after 72 h)to(+)-α-pinene,(-)-α-pinene,(+)-β-pinene,(-)-β-pinene,(R)-(+)-limonene,(S)-(-)-limonene,(1S)-(-)-verbenone,(+)-3-carene,myrcene,tridecane,and(R)-(-)-α-phellandrene by electroantennographic analysis.The reduction in electroantennographic(EAG)responses showed gender differences.In general,female reduced more than male in EAG responses.To different volatiles,the response level of the ds RNA-treated beetles to(+)-α-pinene,(-)-α-pinene,(+)-β-pinene,and(-)-β-pinene were remarkably lower than those of the water-injected controls with a reduction of approximately25.45～88.21%.The response of myrcene and tridecane displayed unconspicuous change with a reduction of about 43%.9.Primer used to clone the Darm Orco was designed based on homology genes of Orco and RACE PCR was used to obtain the full-length c DNA of Darm Orco.The results of sequence analysis showed that Darm Orco shares highly sequence homology with Orco proteins of other insects,transmembrane topologyprediction indicated that Darm Orco has 7transmembrane domains,the same with GPCRs(G protein-coupled receptors),but it adopt a reverse membrane topology—extracellular C-termini and intracellular N-termini.Real-time PCR analysis showed that Darm Orco expressed abundantly in adult stage of D.armandi;by contrast,the expression level in other stages were trace amounts.In different tissues,the expression level of Darm Orco was the highest in the antennae.In order to understand the functional significance of Orco,we injected si RNA of Darm Orco into the conjunctivum between the second and third abdominal segments,and evaluated its expression after si RNA injected for 24 h,48 h and 72 h.The results of q RT-PCR demonstrated that the reduction of m RNA expression level was significant(～80%)in Darm Orco si RNA-treated D.armandi than in water-injected and non-injected controls.The electroantennogram responses of females and males to 11 major volatiles of its host,were also reduced(30～68% for females;16～70% for males)in si RNA-treated D.armandi compared with the controls. |
PDF Full Text Request