Genome-wide Identification Of Genens Encoding Detoxification Metabolic Enzymes And Exploration The Function Of DcitCYP4C4 In Citrus Psyllid,Diaphorina Citri

The Asian citrus psyllid?ACP,Diaphorina citri?are the main vectors of Huanglongbing?HLB?.At present,there is no effective method to control HLB,so the control of D.citri has become an effective measure to control HLB.Among them,chemical prevention is the most effective prevention method,but due to people's abuse of pesticides,not only caused serious environmental pollution,but also made the resistance of D.citri increased recently.A large number of studies have shown that the relationship between insect resistance and detoxification metabolic enzymes.So in order to control D.citri more effectively,it is necessary to understand the molecular mechanism of resistance.In this research,three detoxifying metabolic enzymes of D.citri were identified.QPCR was used to analyze the expression model of detoxification metabolic enzyme genes in different tissues and insecticide induction.The full-length sequence of Dcit CYP4C4 gene was obtained by molecular cloning.RNAi and bioassay were used to determine the importance of DcitCYP4C4 gene in the susceptibility of imidacloprid and chlorpyrifos in D.citri.The results provided the theoretical basis for the importance of P450 gene in the development of resistance in D.citri.The main results of this study are as follows:1 Genomic identification of three detoxifying metabolic enzymes of D.citriThrough blast and phylogenetic analysis,33 P450 genes were divided into 4 clades,including 11 CYP4 clades,8 CYP3 clades,10 CYP2 clades and 4 Mitochondrial clades.There were 11 GST genes,and they were divided into 5 families,including 5 Sigma genes,3Epsilon genes,1 Delta gene,1 Microsomal gene and 1 Omega gene.There were 19 esterase genes,belonging to 6 families,including 9?-esterase?E?genes,4 acetylcholinesterase?J?genes,3 mitochondrial,cytosolic,2 neuroligin?K?genes,1 glutactin?H?gene,1 gliotactin?L?gene and 1 uncharacteristic clades?I?gene.2 Analysis the mRNA expression pattern of detoxification metabolic enzymes in D.citriThe expression patterns of detoxifying metabolic enzyme genes of D.citri were analyzed by qPCR in different tissues and under the induction of imidacloprid and chlorpyrifos LC₂₀.In different tissues,the expression patterns of P450 family genes in the midgut and malpighian tube were basically the same,4 genes of GSTs family were significantly highly expressed in the fat body,2 genes were significantly highly expressed in the midgut and 5 genes were significantly highly expressed in the malpighian tube,and the tissue expression pattern of genes of EST family showed that the expression level of 3 genes in fat body was significantly higher than that of other genes,the expression of 4 genes were significantly higher in midgut,and 5 genes were significantly highly expressed in the malpighian tube.In P450,after imidacloprid induction,6 genes were significantly up-regulated and 5 genes were significantly down-regulated compared with the control,in GSTs,after imidacloprid induction,2 genes were significantly up-regulated and 3 genes were significantly down-regulated compared with the control,in ESTs,after the induction of imidacloprid,3 genes were significantly up-regulated and 2 genes were significantly down-regulated compared with the control.3 Cloning and sequence analysis of DcitCYP4C4 in D.citriAccording to analyse the expression pattern of metabolic enzymes after insecticide induction and different tissues,we found that DcitCYP4C4 gene was extraordinary.The results of cloning and sequence analysis showed that the ORF of DcitCYP4C4 contains 1545nucleotides,encoding 512 amino acids.The predictive coding protein molecular weight of CYP4C4 is 58.995 KDa and the isoelectric point is 8.26.It has five conservative P450 family structure domain:Helix C,Helix I,Helix K and heme-binding region.The cluster analysis showed that it is closed to DmCYP4D8.4 The functional verification of DcitCYP4C4 in D.citriThe DcitCYP4C4 gene of D.citri from Guilin and Jiangxi was silenced by RNAi,and the mortality rate was detected.The results showed that DcitCYP4C4 could be significantly silenced after dsRNA infusion for 24 h,with the silencing efficiency of 62.87%and 63.60%respectively in Guilin and Jiangxi.After interference 12 h,citrus psyllid were treated with imidacloprid?30.603 mg/L?and chlorpyrifos?55.526 mg/L?respectively.The mortality rate was detected 24 h after treatment.The results showed that Dcit CYP4C4 gene was silenced effectively.The mortalities of D.citri in Guilin and Jiangxi were increased 4.84 and 1.40 fold after chlorpyrifos treated 24 h,the sensitivity of imidacloprid in 48 h significantly increased1.25 fold in Guilin strain.It was speculated that Dcit CYP4C4 might play an important role in the insecticide resistance pathway of D.citri.Through comprehensive analysis,this dissertation identified the genes of the detoxification metabolic enzyme family of D.citri and analyzed the expression patterns in different tissues and after insecticide induction.Then we explored the role of the detoxification metabolic enzyme CYP4C4 in the insecticide sensitivity of D.citri,and laid a certain foundation for the molecular research on insecticide resistance of D.citri.