Validation Of Acetylcholinesterases In Pardosa Pseudoannulata And Cloning New Acetylcholinesterase Genes

Acetylcholinesterase(AChE)is one kind of serine hydrolase and mainly distributed at cholinergic synapses,and most gathered in the folds of postsynaptic membrane of motor nerve.It plays an important role in neural signal transmission by hydrolyzing neurotransmitter acetylcholine(ACh)and interrupting the nerve impulses to keep normal transduction of signal molecules.AChE is the target of organophosplate and carbamate insecticides,which irreversibly inhibit the hydrolysis of ACh by phosphorylation or carbamoylation of residues at the active sites,causing the overexcitation of acetylcholinergic receptors(AChRs),disrupting normal nerve conduction,producing the poisoning symptoms and finally leading to the death of insects.Pardosa pseudoannulata is predatory natural enemy with a broad spectrumn which distributes in most of rice fields of Asia and mainly preys on Nilaparvata lugens,one of the most important insect pests on rice.P.pseudoannulata is one of the most vital predatory natural enemies against insect pests in paddy fields,and it is of significance to control rice insect pests because of its large size,big population,wide space niche and high predatory capacity on plant hoppers.Nowadays,the degradation of the ecological environment makes people realize the significance of environmental preservation,and a better exploitation and utilization of nature enemies for pest management has become a social hot spot.The current domestic and foreign researches are mostly focus on toxicological tests of pesticides agaisnt pest and their natural enemy spiders,but there are few studies on the insecticide selectivity between natural enely spiders and insect pests.We first develop a RNAi technique in P.pseudoannulata to test the insecticide sensitivities of PpAChE1 and PpAChE2 to organophosphate and carbamate insecticides.In a previous transcriptone data from our laboratory,several unigenes were also annotated as AChEs and showed high degrees of similarities with AChEs from other spider or insect species which indicates that there would be more ace genes encoding AChEs in P.pseudoannulata.Based on these information,4 putative ace genes(Ppace3,Ppace4,Ppace5,Ppace6)were cloned from P.pseudoannulata,and compared to AChEs from insect species.1.Validaton of insecticide sensivities in PpAChE1 and PpAChE2 by RNAi techinqueIn the previous studies,two acetylcholinesterases,PpAChE1 and PpAChE2,were identified in P.pseudoannulata.In order to understand insecticide sensitivities in two AChEs,a RNAi technique was first developed in this spider.For a suitable microinjection RNAi method,three paralmeters were optimize.The ventral abdomen near the gorad,50nL and 60h was separately chosen as the injection site,optimum injection volume and interference time.With this RNAi method,it was proved that both PpAChE1 and PpAChE2 were the targets of organophosphate insecticides because RNAi agaist each ace gene could significantly influence the senesitivity,but PpAChE1 would be a major one.PpAChE1 was the main target of carbamate insecticides,but PpAChE2 was not an important target of carbamate insecticide,because RNAi against Ppace2 almost did not change sensitivities to carbamate insecticides.2.Cloning and sequence analysis of new acetylcholinesterase genes in P.pseudoannulataThe full sequences for four putative ace genes were cloned by analysis of P.pseudoannulata transcriptome database and RACE technique.These four predicted proteins exhibite the common features for an AChE,including oxyanion hole G118,G119,A201,a choline binding site W84,Conserved active site triad S200,E327,H440,six cysteines putatively forming intrachain disulfide bonds,C67-C94,C254-C265,C402-C521,and the sequence FGESAG flanking serine active site.However,not all important amino acid residues are conserved in all four putative AChEs,such as Trp84 as part of the choline binding site was replaced by Tyr,Glu327 on catalytic triad was replaced by Asp,some amino acids were changed in the sequence FGESAG and the number of the conserved aromatic amino acid residues lining the catalytic gorge are different.These results indicated that,although four new genes were putative ace genes,new functional studies were needed to confirm the conclusion.According to phylogeny relationships and homologous comparison,four new genes were denoted as Pp-ace3,Pp-ace4,Pp-ace5 and Pp-ace6.By comparing the amino acid sequence of four putative P.pseudoannulata AChEs and AChEs from insect species,several amino acid differences at important sites were found.These amino acid differences may importantly contribute to insecticide selectivity between predatory natural enemy spiders and insect pests,which require further studies in future.