| The carmine spider mite,Tetranychus cinnabarinus,as one of the major agricultural pests worldwide,mainly feeds on economically important plants.For many years,because of the intensive use of acaricides and high reproductive potential and extremely short life cycle,this mite has developed resistance against most of the acaricides,and caused huge losses to agricultural production.Fenpropathrin as a kind of commonly used pyrethroids pesticide,was widely used in control of insects and mites.Many studies has shown that T.cinnabarinus,Tetranychus urticae and Panonychus citri have produced a high resistance to it.Cytochrome P450 monooxygenases(P450s),as an important family of detoxification enzymes,participate in the metabolism of agrochemicals in almost all agricultural pests and play important roles in the development of insecticide resistance.To date,there exist few reports about P450 s were involved in the fenpropathrin resistance in T.cinnabarinus,and more focused on the study of biochemical level.Thus,carrying out study on P450 s and its expression regulation system will be conducive to obtaining a comprehensive understanding of the action modes and roles of P450,which can help to further reveal the metabolism resistance mechanism mediated by P450 s in T.cinnabarinus.The main results of this research are as follows: 1.Toxicity of fenpropathrin to SS and FeR strains of T.cinnabarinusThe residual coated vial(RCV)method was adopted to detected the LC50 of fenpropathrin to susceptible strain(SS)and fenpropathrin resistant strain(FeR)of T.cinnabarinus.The LC50 of the SS and FeR strains were 606.98 mg/L and 61581.93 mg/L,respectively.The result showed that the fenpropathrin resistance ratio of FeR strain was 101-fold and reached a higher level of resistance.Synergist analysis showed that PBO could enhance the lethal effect of fenpropathrin on mites.The resistance ratio of FeR strain decreased from 101-to 75-fold with the application of PBO,which meant that the synergist PBO eliminated 26-fold fenpropathrin resistance.These results preliminarily indicated that P450 s played an important role in the metabolism of fenpropathrin in T.cinnabarinus.2.Identification,cloning and analysis of P450 genes and its function related gene involved in fenpropathrin resistance in T.cinnabarinusFrom the DGE and DNA microarray data,we obtained six overexpressed P450 Unigenes in the FeR strain of T.cinnabarinus.Identification of the differences in the levels of expression of these six P450 Unigenes between the SS and FeR strains indicated that all of them were constitutively overexpressed in the FeR strain.The expression of six P450 Unigenes were significantly upregulated 1.96-to 5.92-fold in the FeR strain compared with SS.The result of qPCR analysis also showed that the expression level of CPR in the FeR strain was significantly up-regulated 4.12-fold than that in SS.These results preliminary showed that six overexpressed P450 genes and one CPR gene may involved in fenpropathrin resistance in T.cinnabarinus.Based on the transcriptome data of T.cinnabarinus and the genome of T.urticae,the full lengths of six T.cinnabarinus P450 genes and a CPR gene containing complete open reading frames were cloned and sequenced.According to their sequence information,these six P450 genes were named as CYP384A1,CYP389B1,CYP391A1,CYP392A26,CYP392A28 and CYP392D11 by the P450 nomenclature committee(David R.Nelson).The GenBank accession numbers are KT851973,KF770839,KT851972,KF77083,KT851975 and KT851974,respectively.The lengths of open reading frames(ORFs)ranged from 1488 bp to 1671 bp,and the molecular weights of the predicted proteins ranged from 56.79 kDa to 64.03 kDa.The CPR gene was named as TcCPR,and the GenBank accession numbers is KP710970.TcCPR contained an ORF of 2004 bp and encoded a deduced peptide of 667 amino acid residues with predicted MW of 75.47 kDa.Furthermore,the bioinformatics analysis showed that all of these six P450 proteins were microsomal P450 s and include a series of typical conserved P450 motifs,such as Helix C,Helix I,Helix K,meander region and the heme binding domain.The maximum likelihood tree illustrates the phylogenetic relationship of these six P450 genes with T.urticae's P450 genes.Sequence alignment showed that CYP392A26,CYP392A28 and CYP392D11 belong to Clan 2,CYP384A1 belongs to Clan 3,and CYP389B1 and CYP391A1 belong to Clan 4.The bioinformatics analysis also showed that TcCPR consisted of transmembrane structure,signal peptide and three conserved domains such as FMN,FAD and NADPH binding regions and shared the greatest identity with T.urticae TuCPR.3.Specific expression patterns of P450 genes and TcCPR in different developmental stages and upon fenpropathrin inductionTo investigate the expression patterns of these six P450 genes in different developmental stages,qPCR analysis were performed from eggs,larvae,nymphs and adults of the FeR strain.The result showed that the transcripts of six P450 genes were specificity in different development stage of T.cinnabarinus.To further reveal the relationship between P450 s and fenpropathrin resistance in T.cinnabarinus,the expression of these six P450 genes at 6 h,12 h and 24 h after fenpropathrin treatment were detected.From the results,we can find that there were no significant differences between treatments and control in SS.However,after treatment 6 h,12 h and 24 h with fenpropathrin,transcripts of these six P450 genes increased significantly in the FeR strain.Specific expression showed that these P450 genes were overexpressed and were more inducible when treated with fenpropathrin in the FeR strain compared with SS.These results further indicated that both constitutive and inducible regulation of P450 genes were potentially involved in fenpropathrin resistance in T.cinnabarinus,and the expression of these six P450 genes were more sensitive to fenpropathrin stress in the FeR strain.qPCR analysis also showed that the expression pattern of TcCPR was consistent with most P450 genes in different developmental stages of T.cinnabarinus.The higher expression quantity were observed in larvae,nymphs and adults,while the mRNA level was the lowest in eggs.The expression levels of TcCPR in larvae,nymphs,and adults were 5.40-,4.47-,and 4.75-fold of the expression level in eggs.Moreover,this phenomenon also suggested that a cooperative action existing between P450 s and TcCPR in T.cinnabarinu.The result of fenpropathrin induction experiment revealed that the TcCPR was up-regulated in the FeR strain after treated with fenpropathrin.The relative expression levels of TcCPR in the FeR strain increased to 3.70-,3.95-,and 4.61-fold compared with control after 6 h,12 h,and 24 h of fenpropathrin induction,respectively.However,a much lower level(< 1.50-fold)of induction for TcCPR was detected in SS.These results once again suggested that P450 s,including TcCPR,were more sensitive to fenpropathrin stress in the FeR strain of T.cinnabarinus.4.RNA interference(RNAi)of P450 genes and TcCPR in T.cinnabarinusTo clarify the specific influence of the over-expressed P450 genes on the susceptibility to fenpropathrin in T.cinnabarinus,a RNAi via leaf-disc feeding experiment was performed.The result showed that after feeding of specific P450 dsRNAs(1000 ng/?L),the expression of the target P450 genes(CYP384A1,CYP389B1,CYP391A1,CYP392A26,CYP392A28 and CYP392D11)decreased significantly(55.27% to 74.19%)as compared with the control(water),indicating an effective silencing of P450 s by RNAi in T.cinnabarinus.The specific activities of individual P450 enzymes decreased significantly(1.21-to 2.29-fold)after feeding of their P450 dsRNAs compared with the control(water).Furthermore,after feeding of P450 dsRNAs,we also assessed the susceptibility of FeR mites to fenpropathrin.The mortalities increased significantly,ranging from 8.39% to 15.78%(LC30)and from 7.88% to 17.49%(LC50),after feeding of individual P450 dsRNA to the FeR strain.More interestingly,the qPCR analysis showed that the expression levels of all six P450 genes decreased significantly(35.93% to 65.64%)after feeding with a mixture of all of their P450 dsRNAs compared with the control(water).After feeding a mixture of all six P450 dsRNAs,the P450 activities decreased more significantly(4.0-fold)than when feeding with individual P450 dsRNA,and the mortalities increased by 30.73% and 33.17% when treated with fenpropathrin(LC30 and LC50),which were much higher than those when feeding with individual P450 dsRNA.Thus,these results indicated that all six P450 genes were collaboratively involved in fenpropathrin detoxification in T.cinnabarinus.After RNAi of TcCPR,the expression of TcCPR significantly decreased to 52.5% and 41.0% in SS and FeR strains,respectively.The P450 s activity also decreased more than 4.09-fold in the FeR strain and the mortalities increased 21.60%(LC30)and 26.20%(LC50)significantly after RNAi of TcCPR in the FeR strain.However,there was no significant difference in the susceptibility to fenpropathrin between TcCPR knockdown treatment and controls in SS.What is more,downregulating TcCPR would not impact on P450 genes expressions,and TcCPR is not a regulator of P450 expression.These results indicated that TcCPR can affect the activity of P450 s and the P450/CPR complex could be an important factor involved in fenpropathrin detoxification.5.Prokaryotic expression of P450 genes and TcCPR in T.cinnabarinusProkaryotic expression system(Escherichia coli)was also utilized to investigate the function of the P450 genes and TcCPR.All of six P450 genes and TcCPR gene were successful expressed in E.coli.The results of Western boltting analysis showed that four recombinant P450 proteins(CYP384A1,CYP389B1,CYP392A28 and CYP392D11)and recombinant TcCPR protein were soluble protein,but only recombinant CYP384A1,CYP389B1 and TcCPR proteins showed the activities of enzyme.The specific activity of recombinant TcCPR is 512.69 ± 139.21 nmol/mg pro.min-1(with cytochrome c as the substrate).The PNOD activities of recombinant CYP384A1 and CYP389B1 are 3.74±0.29 nmol/mg pro.min-1 and 0.97±0.11 nmol/mg pro.min-1,respectively(with p-nitroanisole as the substrate).IC50 of fenpropathrin on inhibiting the PNOD activity of recombinant CYP384A1 and CYP389B1 were 181.3±18.8 ?M and 259.3±116.1 ?M.What is more,the metabolism rate of fenpropathrin was 30.83±3.87% when incubated with 10 ?g of recombinant CYP384A1 prorein,and when the amount of recombinant CYP384A1 was doubled,the rate of fenpropathrin depletion increased to 40.75±3.50%.The metabolism rate of fenpropathrin was 22.57±6.88% when incubated with 10 ?g of recombinant CYP389B1 prorein,and when the amount of recombinant CYP389B1 was doubled,the rate of fenpropathrin depletion increased to 30.62±3.16%.These results directly demonstrated that the P450/CPR complex involved in fenpropathrin resistance in T.cinnabarinus.6.Xenobiotic transcription factors regulate the expression of P450 genes involved in fenpropathrin resistance in T.cinnabarinusThe full lengths of six T.cinnabarinus transcription factor genes were verified by gene clone and sequencing.According to their sequence identity,these six transcription factor genes were named as CncC,Maf,HNF4,HR96 and USP(RXR1)and USP(RXR2).The qPCR analysis showed that the expression of CncC,Maf and HNF4 were significantly up-regulated(394.83-,3.03-and 4413.57-fold,respectively)in the FeR strain,but that of HR96,USP(RXR1)and USP(RXR2)showed no significant difference between SS and FeR strains.The knockdown of the expression of CncC in the FeR strain showed a significant decrease in CYP389B1,CYP391A1 and CYP392A28 mRNA levels and activities of P450 s resulting in 12.75%-20.40% mortality increased when the RNAi mites were exposed to the fenpropathrin.The knockdown of Maf also showed a significant decrease in CYP389B1 and CYP392A28 mRNA levels and activities of P450 s with 19.50%-21.10% mortality increased of mites were observed upon fenpropathrin treatment.However,HNF4 knockdown mites did not show significant increase in mortality upon treatment with fenpropathrin.The expression of six up-regulated P450 genes and activities of P450 s also were not affected by HNF4 knockdown.Therefore,in this study,RNAi-based knockdown of possible xenobiotic transcription factors indicated that CncC and its heterodimer partner Maf may control the expression of CYP389B1,CYP391A1 and CYP392A28 in T.cinnabarinus.The luciferase reporter assays were further conducted to determine the regulation of CncC and Maf on the luciferase activity of P450 promoters.we investigated the importance of both CncC and Maf in regulating the promoter region of CYP389B1,CYP391A1 and CYP392A28 in T.cinnabarinus,and found that binding of both CncC and Maf could enhance the luciferase activity significantly than binding of CncC alone in CYP389B1 and CYP392A28.However,the luciferase activity was the highest when binding of CncC alone in CYP391A1.Interestingly,we did not observe any significant changes in the luciferase activity when the P450 promoters were cotransfected with the Maf alone.Therefore,the current results not only confirmed the results of RNAi(the expression of CYP389B1,CYP391A1 and CYP392A28 were regulated by CncC and Maf),but also further clarified the regulatory relationship between transcription factors and P450 genes: the expression of CYP389B1 and CYP392A2 were regulated by CncC or CncC-Maf heterodimers,and the expression of CYP391A1 was regulated by CncC.The involvement of Maf homodimers in regulation of P450 genes may be impossible in T.cinnabarinus. |
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