Resistance Monitoring Of Panonychus Citri To Pyridaben And Identification Of Genes In Response To Pyridaben Stress

Panouychus citri?Mc Gregor?is the world's largest pest mite in citrus industry.The large number occurrences each year seriously damage the quality and yield of citrus,and also affect the development of the citrus industry.Chemical spraying is still the main method for controlling the citrus mite,but it has developed severe resistance to a variety of acaricides.Cytochrome P450s,as a kind of primary metabolism and activation enzymes in insects or in mites,plays an important role in the detoxification metabolism of exogenous substances.Numerous studies have shown that P450s are involved in the detoxification metabolic process of insecticides in insects or in mites.So far,the research on P450s of P.citri is slightly inadequate,and there are few reports on that P450s involved in response to acaricides and in the detoxification of acaricides.The resistance monitoring to pyridaben in different geographical populations of P.citri and grasps the development of its resistance.To identify the P450 genes involved in the formation of pyridaben resistance,a potential target P450 gene was obtained by means of comparative transcriptome analysis.The feeding method was used to deliver siRNA to the mites to study the detoxifying and metabolic ability of P450 gene to pyridaben.Using prokaryotic expression system,soluble and purified P450recombinant protein was successfully expressed and its enzymatic properties were analyzed.The high-performance liquid chromatography?HPLC?was used to detect the correlation between P450s and pyridaben.The purpose of this study was to clarify and analyze the mechanism of P450s-mediated pyridaben resistance in P.citri,and to provide a basis for the proposed control strategy of P.citri in the field.The main contents are as follows:1 Field resistance monitoring to pyridabenThe results of resistance monitoring showed that P.citri in all citrus producing areas has different degrees of resistance to pyridaben and its resistance level has been increasing in recent years.In some areas,such as:Anyue,Nanning,Yuxi and Guilin,the citrus mite develop a very high level of resistance to pyridaben,and its resistance ratios has exceeded 4000 times.In addition,the resistance level showed great regional differences.The resistance level to pydaben of the red mites in citrus-producing areas in southern China?Nanning,Guilin and Yuxi?was generally higher than that in Sichuan and Chongqing.2 Transcriptome sequencing and differential expressed gene analysisIIIThrough the transcriptome sequencing technology,differential expressed gene analysis screened out the P450 genes that were differentially expressed after induction of pyridaben.6280 Unigene were annotated,among of them 226 Unigene differential expressed?84 Unigene were significantly up-regulated,142 Unigene were down-regulated?.Only one differentially expressed P450 gene,CYP4CL2,was up-regulated with 8.8-fold.This gene was used as a target gene to analyze the detoxification metabolism of P.citri to pyridaben.Meanwhile,some genes were up-regulated in other detoxification pathways,like AMP deaminase,acyl-CoA:lysophosphatidylglycerol acyltransferase and sphingosine kinase.3 Analysis of detoxification metabolic function of Panonychus citri CYP4CL2 on pyridabenThe target gene was silenced in mites delivered by si RNA through leaf dish mediation.The 24-hour silencing efficiency was detected by qPCR.After 24 h of RNAi,the expression of CYP4CL2 was significantly down-regulated by 28.7%.After silencing CYP4CL2,the mortality of P.citri was significantly increased by nearly 25%with LC₃₀ of pyridaben.Based on the E.coli prokaryotic expression system,SDS-PAGE and Western blot analysis,the recombinant proteins CYP4CL2 and PcCPR were successfully expressed,and soluble recombinant proteins were obtained after renaturation and purification..Cytochrome C as a substrate,the activity of PcCPR recombinant protein was detected,and its specific activity was 453.129±56.641 nmol/mg pro.min^-1.Similarly,by adding the PcCPR recombinant protein and using p-nitroanisole as a substrate to detect the demethylation activity of the CYP4CL2 recombinant protein,the specific activity was calculated to be 2.905±0.261 nmol/mg pro.min^-1.The metabolism ability of CYP4CL2 toward to pyridaben was investigated in vitro by HPLC.Chromatogram shows that CYP4CL2 protein cannot directly metabolite pyridaben.It is speculated that CYP4CL2 may play a role in pyridaben detoxification through other pathway or to protect P.citri by other metabolic pathways.