| Propylaea japonica belongs to Coleoptera,Coccinellidae,Having a good adaption to environment,they pray on aphids,leafhoppers,plant hoppers,besides,they have the advantages of high rate of fertility and mass raising.P.japonicacan be a promoting natural enemy in the future.In recent years,the study on biology of natural enemy insect,evaluation of field toxicity and ecological characteristics is growing.Many natural enemies were applied to prevent pests in practice.But the study on molecular biology of P.japonica is still not enough.Acetylcholinestrase(AChE)is a serine protease,which mainly exists in neural and neuromuscular junctions.AChE is the target enzyme for organophosphorus and carbamate pesticides,due to the extensive use of pesticides in recent years,a large number of insects on organic phosphorus,amino acid ester and other pesticides produced a different degree of resistance.The Coleoptera ace1 genes have been cloned only in Tribolium castaneum and potato beetles,and the study on molecular mechanism of P.japonica has not been reported so far.In this article,we first clone the ace l gene of sensitive and resistant P.japonica,the sensitive ace1 gene full-length is 2928bp,(GenBank accession No:KP403801),containing a 1938bp open reading frame(ORF),encoding 645 amino acid,and the resistant ace1 gene full length is 2098bp,(GenBank accession No:KT779500),containing a 1938bp open reading frame(ORF),encoding 645 amino acid.By comparing the amino acid sequence of sensitive and resistant P.japonica ace1 gene with other species,we found that P.japonica ace1 homology as high as 85.4%with Tribolium castaneum ace1,and homology as high as 78.8%with Colorado potato beetle,Leptinotarsa decemlineata ace1,and between 63.5-85.4%with other ace1,33.4-39.7%with ace1.The phylogenetic tree showed that Propylaea japonica ace1 clustered into other species of ace1,having a highest relationship with Coleoptera insect.Comparing the ace1 gene amino acid of sensitive and resistant P.japonica,it was found that five amino acid residues had replaced between PJ-S and PJ-R(Phe 358 Ser,Lys 493 Glu,Lle 538 Val,Gly 571 Arg and Thr 576 Ala),.P.japonica susceptible and resistant ace1 gene has a FGESAG(locus 266-271)highly conserved region.and by comparison,we found that in P.japonica sensitive and resistant AChE,the activity center of the catalytic triad,oxyanion hole,choline binding sites,peripheral anionic site(PAS)and portal site animo acids were similar to electric ray,Pj-S ace1 pocket consists of Phe 358 and Phe 360,which consistent with Phe290 and Phe 292 of electric ray,there has a mutations in Pj-R ace1.11 aromatic amino acid is conservative in 14 aromatic amino acid residues.In this paper,we mainly analyze the ace1 relative expression levels of sensitive and resistant P.japonica by the Real-time PCR,and the results suggested that the expression of Pj-R is 5 times of Pj-S,the increase of ace1 expression strengh the insect resistant to pesticides,and the reduce of ace1 expression impair the insect resistant to pesticides.In view of this,we speculate that the reason why ace1 expression in resistant P.japonica higher than sensitive P.japonica may associated with drug resistance.We successfully cloned ace1 gene of resistant and susceptible P.japonica in this paper,and provides a theoretical basis for the study of P.japonica resistant mechanism and drug resistant molecular mechanism.It can also be applied to study the rational use of natural enemies to control pests,and lay the foundation for reducing the use of pesticides. |
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