Cloning And Characterization Of The Acetylcholinesterase Gene From Locusta Migratoria Manilensis

Acetylcholinesterase (AChE,EC 3.1.1.7) is a key hydrolase distributed at cholinergic synapses in vertebrate animals, nematodes, and neural-muscle conjunctions in invertebrate animals. It plays an important role in neural signal transmit by catalyzing the hydrolysis of the neurotransmitter acetylcholine, blocking the exciting effect of acetylcholine on post synapse membrane. The enzyme is the primary target of organophosphate (OP) and carbamate (CA) insecticides, which work as AChE inhibitors.The oriental migratory locust, Locusta migratoria manilensis, is one of the most important agricultural pests in China because of its wide distribution, massive outbreak, and long distance migration. Organophosphate has been the predominant chemical pesticide to control the locust for a long time. The locust has developed pesticide resistance under the selection pressure.Comparative studies of AChEs from susceptible and resistant oriental migratory locust populations showed that AChE purified from the resistant population is 62-, 2.0-, and 1.6-fold less sensitive to chlorpyrifos oxon, demeton-S-methyl, and paraoxon respectively than that from the susceptible population. As in other arthropods, it implies that the less susceptible AChE confers resistance to these pesticides in oriental migratory locust. Although the insensitive AChE is an important mechanism for insecticide resistance in many pest species, the molecular basis of the altered forms of AChE, is unknown for L. migratoria manilensis because nothing is known about its gene sequence.Here we report the results of the cloning of the AChE gene from L. migratoria manilensis using degenerate primers designed according to the conserved region of other AChEs and 3′-end and 5′-end extending with gene specific primers. The function of the AChE was concluded on the basis of RNA interference, malathion sensitivity bioassay, and Pichia pastoris expression.1. Cloning of AChE geneA 263 bp gene fragment was cloned using degenerate primers. Bioinformatics analysis showed that product derived from the fragment shared 93%, 90% and 89% identity with Blattella germanica AChE1, Liposcelis bostrychophila AChE, and Nephotettix cincticeps AChE2 respectively. The remaining sequence was isolated by 3′- and 5′- rapid amplification of cDNA ends (RACE). The whole sequence was deposited in GenBank (accession No. EU231603). L. migratoria manilensis AChE cDNA was 2255 bp and contained a unique open reading frame (ORF) spanning nucleotides 287-1924. The polypeptide deduced from the ORF comprised 546 amino acids. The predicted protein exhibited all the common features for an AChE including:â‘ Conserved active site triad, S151 (S200 in Torpedo), E277 (327) and H391 (440);â‘¡A choline binding site W36 (W84 in Torpedo);â‘¢Five out of the six cysteines putatively forming intrachain disulfide bonds (C26, C205–C218 and C353–C519), lacking of the first cysteine forming the first intrachain disulfide bond;â‘£Twelve out of the fourteen conserved aromatic amino acid residues lining the catalytic gorge (Y70, W84, W114, Y121, Y130, W233, W279, F288, F290, F330, F331, Y334, W 432 and Y442 in Torpedo; W36, W66, Y73, Y82, W184, W232, F240, F242, F280, F281, Y284 and W373 in L. migratoria manilensis);⑤The sequence FGESAG, flanking serine active site, conserved in all cholinesterase.2. The relationship between L. migratoria manilensis AChE and OP resistanceIn vitro prepared double stranded RNA (dsRNA) was injected into locust body cavity at fourth instars. The result of quantitative real-time PCR showed that RNAi could suppress 46.14% the gene expression at the level of transcription, the result of ELISA indicated that RNAi could suppress 47.36% the gene expression at the level of translation, and the specific activity of AChE was suppressed 60% according to the result of activity measurement.In order to detect the effect of AChE activity depression on OP sensitivity, bioassay was done on the 6th day after 1.2μg dsRNA injection. The result of malathion sensitivity bioassay showed that the LD₅₀ of the RNA interfered group was 1.73μg, and that of the control group was 4.34μg. The difference between treatments is significant (p<0.05). The result that AChE gene interference should increase the sensitivity of malathion implied that the AChE is the target of OP pesticide which related to OP pesticide resistance in Locusta migratoria manilensis.3. Expression of L. migratoria manilensis AChEThe expression vector was constructed and transformed into Pichia pastoris. Two Pichia pastoris with high expression level was screened out from the transformants using indoxylacetate as substrate. The expression of the strains could reach 500μg·mL^-1 after 9 days of inducing incubation in methanol at the concentration of 2.5%. The supernatant of the expression medium could be precipitated with ammonium sulfate in 50% saturation, and purified by His-Tag chromatograph. The specific activity of the purified recombinant AChE was 8.1U·mg^-1.The result of substrate specificity showed that the recombinant AChE preferred to acetylthiocholine than butylthiocholine, and the Km value is 67.77μM when acetylthiocholine was used. The enzyme activity could be suppressed 69% and 76% by eserine and BW284C51 at the concentration of 1×10-4 mol·L^-1.