Analysis Of Carboxylesterase Between The Natural Enemy Of Pardosa Pseudoannulata And The Pest Nilaparvata Lugens

Carboxylesterase family belongs to one of the three major metabolic enzyme families and belongs to the multi-functional carboxylate/cholinesterase superfamily,which is widely distributed in plants,insects,mammals and microorganisms.Based on the phylogenetic similarity and different physiological characteristics of carboxylesterase genes,the insect carboxylesterase is divided into nine subfamilies:α-esterase,juvenile hormone esterase(JHE),β-esterase,integument esterase,neuroligin,gliotactin.,neurotactin,glutactin and acetylcholinesterase(AChE).Carboxylesterase plays an important role in the development of pesticide resistance of pests and their natural enemies,and also in the metabolism of various exogenous and endogenous esters.Here,the genomic information of Nilaparvata lugens and Pardosa pseudoannulata were analyzed to obtain their respective carboxylesterase genes,which were classified and identified by tissue expression pattern.In the study,we found that there are multiple AChEs in the Pardosa pseudoannulata,which has a greater variety of AChE than any other species to date.Hence,we analyzed it in detail.1.Classification and expression patterns of carboxylesterase in Nilaparvata lugensThe widespread and extensive application of insecticides have promoted the development of resistance in the Nilaparvata lugens.To better understand the underlying molecular mechanisms of metabolic resistance to insecticides,it is necessary to figure out the role of carboxylesterases in the detoxification process of Nilaparvata lugens.Based on the Nilaparvata lugens genome database and the GenBank sequence,26 carboxylanase genes were found.According to the tissue expression profiles of important functional genes,several genes of α-esterase,β-esterase and integument-esterase were highly expressed in midgut,fat body and epidermis.It is suggest that the carboxylesterases of Nilaparvata lugens is of great significance in detoxification metabolism.The present study may be beneficial for us to further understand the role in its insecticide resistance development at the molecular level.2.Classification and expression patterns of carboxylesterase in the wolf spider,Pardosa pseudoannulata Carboxylesterase plays an essential part in the detoxification and metabolism of exogenous substances such as food in insects,mites and spiders.Based on the genome and transcriptome database of the Pardosa pseudoannulata,we identified 28 carboxylesterase genes.We classified them and found that there are no α-esterase,JHE,and glutactin in the Pardosa pseudoannulata.Besides,there are eight AChEs with the catalytic function in neurodevelopment,only one β-esterase and two integumentesterase as well.According to the sequence comparison analysis of these 8 AChEs(4 have been published,and 4 new have been found newly)and mRNA expression levels of 8 AChEs in different tissues of the Pardosa pseudoannulata were determined,and it suggested that the expression levels of ppace1-8 in various tissues were significantly different,with typical tissue specificity.A comparative analysis of the difference in carboxylesterase between insects and arachnids can help us better analyze the selective mechanism of insecticides between natural enemies and insects.Thus we can utilize and protect natural enemies and effectively control pests in a further and deep way.3.Characterization of the fifth putative acetylcholinesterase in the wolf spider,Pardosa pseudoannulataAcetylcholinesterase(AChE)is an important neurotransmitter hydrolase in invertebrate and vertebrate nervous systems.In invertebrates,AChEs is highly diverse and has various functions,including the "classical" functions that terminate synaptic transmission and "non-classical" functions.Based on the four AChEs identified and published,one new putative AChE(AChE5)is studied in this chapter.Sequence analysis showed that AChE5 encoding gene contained most conserved motifs of AChE family.Compared with the published AChEs,the recombinant protein expressed by Sf9 cells in vitro showed significant differences in biochemical characteristics.The comparison of Michaelis-Menten constant(Km)of PpAChE5 hydrolyzed substrates shows that the difference in Km of PpAChE5 hydrolyzed three substrates is not significant,Vmax of PpAChE5 hydrolyzed ATC was greater than that of BTC and PTC.By comparing the catalytic efficiency(Vmax/Km),it was found that PpAChE5 had the highest catalytic efficiency for substrate ATC,indicating that PpAChE5 was obviously biased against ATC.Further inhibitor assay showed that AChE5 was highly sensitive to AChE specific inhibitor BW284C51,but not to butylcholinesterase specific inhibitor ISO-OMPA.The study of AChE will help to understand the evolution and complexity of AChEs in invertebrates.