Discovery Of Genes Related To Digestive Enzymes And Detoxifying Enzymes From Grapholitha Molesta (Busck) Midgut

Grapholitha molesta?Busck?is a world fruit pest that can harm the tender tip and the fruit of many kinds of Rosaceae fruit trees.Seasonal transfer to host plants is the main reason for a G.molesta outbreak.G.molesta has killed natural enemies has survived through environmental pollution and pesticide residue and has generated heavy resistance to insecticides from excessive reliance on chemical pesticides.It is vital to obtain efficient and environmental methods to treat G.molesta.The midgut,as the main organ of insect digestion and detoxification metabolism,plays an important role for insects in feeding and digestion of different host plants,growth and development,and defense against exogenous toxic substances.In this study,high-throughput sequencing technology was used to analyze the transcriptome of the midgut of Grapholitha molesta larvae,and highly expressed digestive enzymes and detoxification enzyme-related genes were obtained.The expression of digestive enzymes and detoxification enzymes in the midgut of different ages was analyzed.This study lays a certain theoretical foundation for the biological control of pests with midgut digestive enzymes and detoxification enzymes as targets.The results are as follows:?1?A total of 96,419 unigenes in the larval midgut transcriptome of G.molesta were obtained,and 57,300 unigenes were annotated in the public databases.In the GO classification,there were 55 functional areas of three major biological processes,cell components and molecular functions.Among them,the most annotated to the cell process,cell and cell components and binding functions.The results of KOG showed that 10,090sequences were classified into 25 gene families,with the most annotated to the general function.In the KEGG database,10,250 unigenes were annotated to 232 metabolic pathways,and the number of genes annotated to ribosomes was the highest.In addition,using MISH software,12,690 SSR loci were searched in 10,600 unigenes,and six SSR loci were developed by PCR.?2?A high expression gene was screened to obtain 2735 highly expressed genes.GO and KEGG enrichment analysis of highly expressed genes revealed that there were 44 GO terms in the GO enrichment analysis,with metabolic process and cellular process having the highest number of annotated genes,followed by binding and catalytic activity.In the KEGG enrichment analysis,the most abundant genes in the five major biochemical metabolic pathways were translation and folding,sorting and degradation.?3?Four kinds of digestive enzyme genes with high expression were obtained.There were 14 trypsin genes,such as CL-10148.17450,CL-10148.18775,CL-10148.21088,CL-10148.17877 and CL-10148.22705.There were five chymotrypsin gene bases,namely CL-10148.19112,CL-10148.19209,CL-10148.19362,CL-10148.20037and CL-10148.21371.There were 14 carboxypeptidase genes,such as CL-10148.11888,CL-10148.15725,CL-10148.19922,CL-10148.20594,and CL-10148.16225.There were20 aminopeptidase genes,such as CL-10148.17557,CL-10148.21926,CL-10148.19992,CL-10148.19118 and CL-10148.19035.The expression levels of trypsin genes 1 and 2,carboxypeptidase genes 1 and 2,aminopeptidase genes 1 and 2 were different in the midgut of G.molesta larvae at different ages by real-time PCR.The levels increased as the age increased,and then decreased,the highest expression at the 4^th instar,followed by the 3^rdd instar.?4?There were three genes for high expression of detoxification enzymes,and 17genes for glutathione S-transferase,such as CL-10148.18663,CL-10148.19812,CL-10148.20548,CL-10148.20001,CL-10148.20002,and the like.There were 17 P450genes,such as CL-10148.18049,CL-10148.19051,CL-10148.21071,CL-10148.20009,CL-10148.21072 and the like.There were 13 carboxylesterase genes such as CL-10148.9606,CL-10148.14567,CL-10148.17622,CL-10148.19098,CL-10148.19910and the like.The expression levels of glutathione S-transferase genes 1 and 2,P450 genes 1and 2 and carboxylesterase genes 1 and 2 in the midgut of G.molesta larvae of different ages were detected by real-time PCR.The levels increased as the age increased,and then decreased,the highest expression at the 4^th instar,followed by the 3^rd instar.