Molecular Characterization And Functional Analysis Of Acetylcholinesterase Genes From Brown Citrus Aphid,Aphis(Toxotera)citricidus(kirkaldy)

The brown citrus aphid,Aphis(Toxoptera)citricidus(Kirkaldy),is considered to be one of the economically damaging and widespread pests in the world.In this study,to better understand the underlying molecular mechanism of acetylcholinesterase(AChE)in A.citricidus,a series of experiments employing biochemical and molecular approaches were conducted.Based on the data of the transcriptome of the brown citrus aphid,two ace genes were screened by bioinformatics.We further sequenced,phylogenetically analyzed,and characterized the stage-and tagmata-dependent expression patterns of the two ace genes,Tcace1 and Tcace2.To evaluate their possible roles in responding to cholinergic and non-cholinergic pesticides,baculovirus expression systems and RNA interference were used to overexpress and knockdown these two gene s respectively.The main results are as follows:1.Sequence and phylogenetic analysisBoth Tcace1 and Tcace2 were sequenced from A.citricidus.Tcace1 cDNA contains a 2,031 bp open reading frame,283-bp-long 5’-UTR and 273-bp-long 3’-UTR.Tcace2 cDNA contains a 1,995-bp open reading frame,163-bp-long 5’-UTR and 57-bp-long 3’-UTR.The Tcace1 and Tcace2 cDNAs(GenBank accession number: KP723526,KP723527)encoded proteins of 676 and 664 amino acid residues,respectively.Both Tcace1 and Tcace2 contained all the highly conserved motifs for maintenance of AChE and all major structural and functional motifs,including six cysteine residues forming three intramolecular disulphide bridges,choline binding sites,acyl pocket,oxyanion hole,catalytic triads,and a number of aromatic residues lining the catalytic gorge.Moreover,both TcAChE1 and TcAChE2 have signal peptides.The phylogenetic trees revealed the evolutionary relationship among the two AChEs of A.citricidus along with other AChEs.The two AChEs from A.citricidus belong to two clusters.In addition,the two AChEs were the most homologous to that of the Acyrthosiphon pisum and Aphis gossypii.2.Stage-and tagmata-dependent expression patterns of Tcace1 and Tcace2The analysis of expression patterns of Tcace1 and Tcace2 showed that both the two genes were detectable in all body sections and developmental stages examined.The expression level from the nymph to the adult is showing a gradual increase in the trend,and expression is slightly higher in the apterous aphid than the winged aphid,and the two genes showed high expression in the head.3.Functional analysis of Tcace1 and Tcace2 based on RNAiRNAi for Tcace1 and Tcace2 was performed to investigate the possible involvements in cholinergic and non-cholinergic functions.RT-qPCR analysis showed that there were significantly decreased transcript levels of Tcace1(55%)and of Tcace2(50%)at 48 h after the indirect feeding of dsRNA as compared with those in the dsGFP-fed aphids(P<0.05),suggesting that 48 h was an effective silencing point of these two genes after dsRNA feeding.And there is no mutual interference and off-target effect.Within 48 h,the mortality of aphids treated with dsRNA of Tcace1 increased significantly(P<0.05).After 48 h,the susceptibility of A.citricidus to the inhibitor was measured.The mortality of A.citricidus was significantly increased after 24 h of treatment with lethal concentration of malathion and carbaryl LC10-LC20(P<0.05),and the sensitivity showed significant changes compared with the control group.When treated with malthion after 24 h,the mortalities were 74% and 52% in dsTcace1-and dsTcace2-fed aphids respectively.In another treatment with carbaryl,the mortalities were 84% and 59% in dsTcace1-and dsTcace2-fed aphids respectively.The mortalities,treated with these two pesticides to dsTcace1-and dsTcace2-fed respectively,showed significant difference between two genes(P<0.05).The activity of AChE was detected after 48 h dsRNA feeding.The residual activity was 61% when knockdown Tcace1,73% when knockdown Tcace2 gene.AChE activity was significantly decreased compared with control(P<0.05).These results indicate that the Tcace1 and Tcace2 genes play an important role in cholinergic and non-cholinergic functions,moreover,the Tcace1 gene plays a more dominant role.4.Functional expression of recombinant enzymesEnzymatic assay of the recombinant TcAChE protein was measured in vitro to evaluate whether there was AChE specific activity.The activity of AChE in Sf9 cells expressing TcAChE1 and TcAChE2 proteins was significantly higher(P<0.05),AChEs are more affinity to the three substrates and have higher enzymatic response rates.The results showed that cells expressing TcAChE1 were more affinity for the three substrates than that of TcAChE2.Detection the bimolecular constant of the expressed protein showed that TcAChE1 protein had higher bimolecular ratio than that of TcAChE2 protein,namely,TcAChE1 was more sensitive to the inhibitor.It was further confirmed that TcAChE1 was the predominant form in mediating nerve conduction and resistance in A.citricidus.In conclusion,the two ace genes were identified based on transprotome data of A.citricidus.The sequence characteristics,phylogenetic relationships,temporal and spatial expression patterns of the two ace genes were analyzed comprehensively.Heterologous expression demonstrated that TcAChE1 showed a higher affinity to substrates than TcAChE2 and it was more sensitive to the ten inhibitors.Furthermore,RNAi-mediated knockdown of each of these two genes significantly increased malathion and carbaryl susceptibility.Taken together,this study revealed Tcace1 and Tcace2 genes play an important role in cholinergic and non-cholinergic functions,and the Tcace1 gene plays a more dominant role.