AChE1Gene Polymorphisms Of The Bird Cherry-oat Aphid, Rhopalosiphum Padi (Hemiptera: Aphididae) From The Field Populations In Different Regions Of China

Acetylcholinesterase (AChE) is an important enzyme in nervous system. It can hydrolyzethe neurotransmitter acetylcholine at the postsynaptic membrane and terminate nerveimpulses causing by chemical signals. AChE is the target of organophosphate and carbamateinsecticides. AChE gene mutation can alter the structure of the enzyme, decrease the AChEsentitivity to insticides, furthermore, the resistance of pest insect to pesticides and AChEmutation has a close relationship.Rhopalosiphum padi is an important pest of wheat in China, and this pest had developedresistance to many insecticides including organophosphates and carbamates. In the currentresearch, we sampled16R. padi field populations from different regions of China andamplified the full-length of AChE1from15samples of each population. Fifteen samples ofthe susceptible laboratory strain were used for comparison. We aligned the obtainednucleotide and amino acid sequences of AChE1, and analyzed the mutations of each sequence.The average number of nucleotide differences (K), nucleotide diversity index (Pi), haplotypediversity (Hd) of different field populations was also analyzed. We estimated the secondaryand tertiary structure of mutated AChE1. The results are as follows:The AChE1gene of255individuals of R. padi from16field populations and thelaboratory susceptible population were amplified. The full-length of AChE1gene was2306bp, which encoded676amino acids. We found49nucleotide polymorphisms in all acquiredsequences, and of which27sites were nonsense mutations. No mutations were found insamples from susceptible strain. Mutation sites of nucleotide mainly located between400bpand1500bp, especially be abundant between400bp and700bp site. In total of46transitionsites were found in field populations, and three transversion sites were found. Thetransversion mainly occured in SXH, HBZ and HBW populations.Twenty-two AChE1nucleotide mutation sites caused14amino acid mutations werefound in R. padi field populations. The main mutations included aspartate (D) to glycine (G),valine (V) to alanine (R), and lysine (K) to arginine (R). Some field populations had the same mutations, such as the mutation D83G and K494R in SDT, SDZ and GZG population, themutation W334R and K441E in HBW, SDZ, SDT population, the mutation R251W andK511R in GSL, HEB and SXH population, and the mutation V295A and D383G in SXH andAHC population. Population GZG, SDT, SDZ had the same mutations, while population SXT,HEB, GSL shared the same mutations. Some unique population was found in HBZ, SAX,SDH and HBW population, respectively.We found an alternative splicing in individuals from nine field populations. Thealternative splicing occurred near the5’end of the AChE1coding region, resulting the deletionof “ATCCGAATTTAG” located at225bp-236bp and the deletion of amino acids “RSEF”.This was the first time that AChE1alternative splicing was found in R. padi filed populations.The Sable online server was used to analyze the secondary structure of the alternative splicingin R. padi AChE1gene, which showed a transmembrane region in the initial position of thecoding region was caused by the deletion. We did not find any mutations at metabolic triad inall the22amino acid mutation site, however, some mutations located in aromatic amino acidwere found. Three mutations, which included the D383G mutation near to the F388of acylpocket, the K441E mutation near to metabolic triad E427, and the W334R mutation of anaromatic amino, may have important effect on the resistance of R. padi to pesticides,With the ClustalX2.1software to align R. padi AChE1in field population and13AChEsequences from different species, we constructed the neighbouring-joining tree of insectAChE sequences. All the insect AChE sequences were divided into two clades in the obtainedtree, with all the AChE1sequences in one clade and all AChE2in another one.