| Asian citrus psyllid(ACP),Diaphorina citri,is an important agricultural pest.It is the main vector of Huanglongbing(HLB),which seriously restricts the sustainable development of citrus industry worldwide.ACP suppression with chemical insecticides is currently one of the most effective strategies for HLB management because there is no useful method to cure HLB.However,it is difficult to prevent ACP effectively due to its strong environmental adaptability,rapid development of insecticide resistance and gradual northward expansion of ACP.Insect odorant-binding proteins(OBPs)serve as the key carrier to connect the external environment and olfactory receptor neurons.They play primary roles in insect olfactory signal transduction process,which are deemed to the most promising pest control target.In the current study,we focused on the physiological functions of OBPs in D.citri based on the analysis of the spatiotemporal expression profiles of nine OBP genes.Furthermore,the Escherichia coli prokaryotic expression system,in vitro fluorescence binding assay,antennae electrophysiology,bioassay,RNA interference,behavioral analysis and molecular docking were employed to determine the physiological functions of OBP7 and OBP2 in chemoreception and imidacloprid susceptibility.Our results provided a theoretical foundation for further developing new control agents for D.citri management.The main results were as follows:1 Identification of the OBP genes in D.citri and their spatiotemporal expression profilesMultiple sequence alignment and phylogenetic analysis showed that nine OBPs can be divided into two types.OBP2 is a Plus-C OBP,and the remaining eight OBPs are Classical OBPs.The expression profiles of nine OBP genes at different developmental stages were determined using RT-q PCR.It was found that the expressions of OBP4 and OBP5 were low in the adult stage,while the transcriptional levels of the other seven OBPs were significantly increased in the adult stage.Among of them,OBP3 and OBP6had the most obvious increase.In addition,the expressions of nine OBPs were similar in female and male adult.Based on the expression profiles of OBP genes in different tissues and body segments,the nine OBPs can be divided into three categories.Five OBP genes(OBP3,6-9)were highly expressed in the head,suggesting that they mainly involved in the chemoreception in D.citri.OBP2,OBP4 and OBP5 had a higer expression levels in leg,external genitals and wing.OBP1 had the highest expression in fat body.2 Heterologous expression and in vitro odorant binding analysis of OBPs in D.citri Using Eescherichia coli prokaryotic expression system,five OBPs recombinant proteins were successfully induced and purified in vitro with molecular weights ranging from 19.4 to 21.6 k Da.All of the five OBPs were supernatant soluble proteins and Western blot further verified that the recombinant proteins obtained were specific OBPs.Twelve plant volatiles expressed at high levels in host plants were selected and the interaction between odorants and OBPs was analyzed using microscale thermophoresis technology.As the results indicated,OBP6 can bind to multiple ligands with broad and high binding affinities(K_d<10μM).OBP7 and OBP8 exhibit relatively wide binding activities,ranged from 0.44-57μM and 2-35μM,respectively.However,OBP9 weakly bound to the tested ligands(K_d>20μM),and OBP3 only bound to two alcohols with high affinities(K_d<10μM).3 OBP7 participating in the response of host plant volatiles in D.citriElectrophysiological results showed that the most host volatiles can trigger the electroantennogram response of D.citri in a concentration-dependent manner,and the amplitude intensity ranged from-0.05 to-0.35 m V.Wherein,the host volatiles including citral,linalool,R-(+)-limonene,and methyl salicylate,which could trigger high EAG reactions of D.citri,also had significant attractively behavioral effects on D.citri adults.Besides,the transcriptional levels of OBP3 and OBP7 were both found to change significantly when induced by these host volatiles.Furthermore,RNAi suppression of the m RNA expression of OBP7 resulted in a significant reduction of EAG activity and the adult D.citri behavioral responses to tested volatiles.Molecular docking analysis showed that most of the binding cavity of OBP7 was composed of hydrophobic amino acids.In addition,a few hydrophilic amino acids in the cavity may interact with ligands to promote binding formation.Especially,Arg107 of OBP7 was a key binding site,which can interact with various ligands to form hydrogen bonds.4 OBP2 in regulating the susceptibility of D.citri to imidaclopridThe expression of OBP2 was significantly increased under imidacloprid induction,suggesting that this gene may participate in regulating the susceptibility of D.citri to imidacloprid rather than chemoreception.Furthermore,ds RNA mediated RNAi of the expression of OBP2 led to a significantly increased susceptibility of D.citri adult to imidacloprid.The purified recombinant protein of OBP2 expressed in E.coli showed a strong in vitro binding capacity to imidacloprid molecule by using MST technology.Additionally,molecular docking revealed that the binding interaction may be easily to be promoted by the hydrogen bonds between hydrophilic residue Ser6 and Lys221 of OBP2 with imidacloprid molecule.In summary,this dissertation analyzed in vitro odorant binding profiles of OBPshighly expressed in the head of D.citri.The results revealed that OBP7 plays a crucial role in the primary stage of olfactory response to host volatiles in D.citri.Meanwhile,OBP2 could bind to imidacloprid molecule with high affinity,effectively sequester and mask the toxicity of imidacloprid molecule to D.citri,reducing the susceptibility of D.citri to imidacloprid.The results not only enriched our understanding of the physiological function of insect OBPs,but also provided potential molecular marker for the field resistance monitor and management of D.citri. |
