Study On The Expression Of Cytochrome P450 Genes Of Grapholita Molesta In Response To Metabolic Insecticides

Cytochrome P450 has been proven to participate in insect metabolism of endogenous secondary metabolites and exogenous toxic substances such as pesticides.There are many types and numbers,and different P450 genes play different roles.In this study,three different types of insecticides,abamectin,imidacloprid and lambda-cyhalothrin,which are commonly used in orchards,were used to treat healthy adult Grapholita molesta(Busck),and screened the differentially expressed cytochrome P450 genes.Analysis of expressed genes to clarify their temporal and spatial expression patterns and lay the foundation for further analysis of their functions.The main findings are as follows:1.As a result of transcriptome sequencing,101,873 sequences were obtained,50,672 CDS were detected,15,478 SSRs were also detected in 12,989 Unigenes,and 9,124 Unigenes encoding transcription factors were predicted.Among the data obtained from the sequencing results,42,247 Unigenes are annotated into the KOG function,which are divided into 25 categories.In the NR annotation results,321 sequences related to "Cytochrome P450" were searched,and 86 cytochrome P450 gene related sequences were screened by Blast comparison,which belonged to CYP2,CYP3,CYP4 and mitochondrial clusters.2.After treatment with abamectin,imidacloprid and lambda-cyhalothrin,four sequences showed a high expression trend.The selected genes were cloned and sequenced and submitted to the naming committee for naming,respectively CYP4L57,CYP428A1,CYP9A114,CYP9A203.3.Ge Norm analysis shows that the most suitable internal reference for different developmental stages are RPL13,18 s r RNA and β-tubulin three internal reference genes for simultaneous use.After treatment with different insecticides,the most suitable internal reference gene obtained by Norm Finder is β-tubulin.4.Combining the 8 P450 genes obtained in the previous stage,study the temporal and spatial expression of12 P450 genes.Expression studies at different developmental stages showed that CYP6AN35,CYP4L57,and CYP9A114 had the highest expression in adults,CYP6AB112-CYP6AB117 had the highest expression in 3rd instar larvae,CYP6B74 had higher expression in 4th instar larvae,and CYP428A1,CYP9A203 had the highest expression in 5th instar larvae.Expression studies in different tissues of adult worms showed that CYP6AN35,CYP4L57 and CYP428A1 were highly expressed in the head and CYP6AN35 only had the highest expression in the head.Except for CYP9A114,CYP6AN35 and CYP428A1,the other nine P450 genes were all highly expressed in the midgut,CYP6AB112,CYP6AB114,CYP6AB115,CYP428A1,CYP9A114 are highly expressed in Markovian ducts,CYP6AB112,CYP6AB113,CYP6AB114 are higher in testes,and CYP6AB115,CYP4L57,CYP428A1,CYP9A114 and CYP9A114 are higher in ovaries.5.Using ds RNA of cytochrome P450 gene synthesized in vitro to study its interference efficiency.Through the expression analysis of the ds RNA fed at different times and different doses,the expression level of G.molesta was not significantly different.The results showed that the feeding method was not effective for the lepidopteran insect G.molesta.