| The citrus red mite, Panonychus citri (McGregor)(Acari:Tetranychidae), is re-garded as the major citrus mite in China. The outbreak of this pest and injury to the plants caused by mite feeding can affect citrus harvest quantity and quality. Control of this mite depends largely on spraying of acaricides. However, frequent acaricides exposure combined with several biology characteristics of this mite (such as high fecundity, short life cycle), facilitate the development of strong resistance in this species to commonly used acaricides. To date, few studies have been attributed to the resistance mechanisms in P. citri, which mostly focus on the biochemical analysis and the identification of resistance genes. As a class of key detoxification enzymes in insects/mites, cytochrome P450monooxygenases (P450s) can metabolize a wide range of endogenous and exogenous chemical substances. Many studies have suggested that P450s in ticks and mites are involved in the resistance to many acaricides/insecticides, including pyrethroids, organophosphates (OPs), Mitochondrial Electron Transport Inhibitors (METI’s), tetronic acids, as well as biological pesticides. Researches around world have been focused particularly on cytochrome P450, while the important P450redox partners NADPH cytochrome P450reductase (CPR) and cytochrome b5are often ignored. Thus, carrying out study on P450and its redox system will be conducive to obtaining a comprehensive understanding of the action modes and roles of P450, which can help to reveal the metabolism resistance mechanism mediated by P450s.Therefore, our study was aimed at the response pattern of P. citri P450s under the stress of xenobiotics. At the beginning, we conducted the monitoring of resistance to four acaricides in P. citri collected from different citrus orchards in Chongqing and compared the activities of three major detoxification enzymes. As a result, cytochrome P450was chosen as the focus of our research. Secondly, we generated a cDNA library containing four different developmental stages of P. citri and identified a number of detoxification enzyme genes, which would provide abundant information for the study on the molecular resistance mechanisms of P. citri. Based on the transcriptome data, we amplified several full-lengths P450s, as well as CPR and CYB5genes. In addition, the expression reaction of P450, CPR and CYB5genes under different chemical stress was further examined. Finally, the eukaryotic expression system of P450genes in P. citri was established preliminarily, which would contribute to the functional verification of cytochrome P450system. The main results are as follows: 1Monitoring of resistance to four acaricides in different field populations of Chongqing and comparison of the activities of major detoxification enzymes1.1Investigation of susceptibility to four acaricides of different field populations of P. citriA modified leaf-dip bioassay method was used to measure the toxicity of four acaricides (abamectin, azocyclotin, pyridaben, spirodiclofen) on four P. citri popula-tions (Beibei, Bishan, Wulong, Zhongxian) sampled from Chongqing. The result showed that abamectin exhibited the highest acaricidal activity among these four acaricides, while the four field populations showed a less susceptibility to spirodiclofen and pyridaben. However, the citrus red mites in Chongqing showed a highest level of resistance to azocyclotin. We learned from the monitoring data for abamectin that, the Bishan population was more susceptible to this acaricide than other three populations. Zhongxian and Wulong populations showed greater tolerance to abamectin and their LC50values were11-and12-folds higher than that of Bishan population. The resistance levels of four populations to azocyclotin were closed and ranged from209.9to379.9mg/L. Samples collected from Zhongxian showed the lowest resistance level to azocyclotin and Bishan population was more tolerant than other populations. Beibei population showed the highest resistance to pyridaben with a LC50value of25.5mg/L, which was4-fold higher than that of the most sensitive population (Bishan). The resistance levels of four populations to spirodiclofen showed great variability and the LC50values were arranged in descending order as follows:Bishan population, Zhongxian population, Wulong population and Beibei population.1.2Comparison of the activities of three major detoxification enzymes1.2.1Comparison of the major detoxification enzymes activities in different developmental stagesWe assayed and compared the P450s, GSTs and CarEs activities in eggs, larvae, nymphs, and adults though a96-well microplate reader. The results suggested that the highest specific activity of P450s was observed in larvae, which was2.66-,2.70-and16.02-folds significantly higher than that in adults, nymphs and eggs. GSTs activity in adults and nymphs were much higher than that in other developmental stages, and the specific activity of GSTs was lowest in eggs. The CarEs specific activity in four stages ranged from0.46to13.60nmol/min/μg, and the value was largest in larva stage and least in egg stage, respectively. In summary, the results indicated that P450s and CarEs might play important roles in metabolism of xneobiotic during the larva stage, while GSTs was involved in protection against environmental stress during the nymph and adult stages.1.2.2Comparison of the major detoxification enzymes activities of different field populationsThe specific activities of P450s was highest in Beibei population, while GSTs and CarEs activities were both highest in Zhongxian population. However, there was no significant correlation between the activities of detoxification enzymes and the re-sistance levels to different acaricides, which might be caused by different background of pesticide application, targets of various acaricides and genetic structures of four populations.1.2.3Comparison of the activities of major detoxification enzymes after xenobiotics treatmentsBased on the bioassay, the mites were treated with LC30of five acaricides (abamectin, azocyclotin, fenpropathrin, pyridaben and spirodiclofen) and one kind of volatile secondary metabolites from citrus (citral) for24h. P450s activities increased after exposure to different chemicals, and reached the maximum value when treated by spirodiclofen. Except abamectin, other five different xenobiotics could significantly induce the GSTs activities. Similarly, GSTs activity was also the highest after treatment with spirodiclofen. However, when stimulated by spirodiclofen, CarEs activity was significantly lower than that of control, while the activities stayed still after exposure to other acaricides and citral. Compared to CarEs, P450s and GSTs could respond to the stress of xenobiotics more quickly and sensitively, which indicated that these two enzymes might take part in the metabolism process of the above xenobiotics.1.2.4Comparison of the major detoxification enzymes activities induced by sodium barbiturateAfter induction with19.96mg/L sodium barbiturate for24h, the activities of both P450s and GSTs increased, and CarEs activity was significantly inhibited by this chemical. 2Transcriptome analysis of P. citri and identification of detoxification enzymes genes2.1Generation and analysis of the transcriptome database of P. citriIn order to obtain a P. citri comprehensive transcriptome, the cDNA library was constructed using the mixture of RNA extracted from four developmental stages and generated by Illumina Hi seqTM2000. As a result, a total of51,214,018clean reads were generated and further assembled into65,149Unigenes. Using BLASTX with a cut-off E-value of10-5, there were32,535Unigenes successfully annotated in nr,25,813in Swiss-Prot,22,725in KEGG,15,412in COG and10,421in GO databases, respectively. The homology analysis of the Unigenes annotated in nr suggested that, P. citri Unigenes had higher similarity to Insecta than other classes;9%of the mite Unigenes showed the highest homology to sequences from Tribolium castaneum, followed by Harpegnathos saltator (6%) and Camponotus floridanus (5%). Additionally,1.7%of the annotated Unigenes were best matched to genes from Ixodes scapularis, which also belonged to Arachnida.The result of GO classification showed that10,421Unigenes which were annotated in nr were divided into three categories and50functional groups. The most abundant group was "cell"(7,035Unigenes) and the smallest group was "channel regulator activities"(2Unigenes). A total of15,412Unigenes annotated in COG were classified into25families. The largest family "general function prediction" contained3,642Unigenes, while the smallest group was "nuclear structure"(11Unigenes).2.2Identification of genes encoding detoxification enzymes and analysis of metabolic pathwaysThere were79P450Unigenes identified from the nr annotation of the P. citri transcriptome. Among these P450-related unique transcripts,12,9,26and7Unigenes were divided to CYP2, CYP3, CYP4and CYP6families, respectively. The remaining25Unigenes belonged to other families, including CYP9, CYP18, CYP314and Mitochondrial CYP family. All the P450Unigenes were screened against KEGG database and58were found to be involved in16pathways. We also identified24GSTs Unigenes from nr annotation of the P. citri transcriptome, which were assigned to the delta, kappa, mu, omega, sigma, theta and zeta families. In the KEGG annotation database,19GSTs Unigenes involved in10pathways were detected. A total of13 sequences encoding CarE were identified from the nr annotation results of P. citri transcriptome, among which there were four, three and one Unigenes were divided into Clade A, Clade B and Clade D, respectively. Only four CarE Unigenes could be annotated in KEGG database and involved in one pathway.3Full-length molecular cloning and sequences analysis of P450and its relative genes from P. citriBased on the transcriptome data,11novel P450genes were cloned from P. citri using the RT-PCR and RACE techniques. All the full-length cDNA sequences were deposited in GenBank and submitted to the P450nomeclature committee. The P450names and the corresponding GenBank accession numbers were as follows:CYP4CL2(JQ690089), CYP384A2(KC201279), CYP385C7(KJ173877), CYP385C8(KJ173878), CYP389D1(KC201278), CYP392A22(KC201275), CYP392A23(KC201276), CYP392A24(KC201277), CYP392A25(KJ173880), CYP392B6(KJ173879), CYP392F1(KC201274). The P450cDNAs contained opening reading frames (ORFs) of1,494-1,758bp that encoding497-585amino acid residues. Furthermore, the bioinformatics software such as ProParam, TMHMM and SignalP3.0were used to analyze the components of protein, physicochemical characteristics, transmembrane structure and signal peptide. As a result, the11P450proteins were considered to be microsomal P450s, which would play important roles in the detoxification of xenobiotics. Protein sequence alignments revealed the typical conserved P450motifs, including the heme binding domain, Helix C, Helix I, Helix K and the meander region. Moreover, the CYP4signature motif EVDTFMFA/GHDTT was present at the amino acid sequence of CYP4CL2.We compared the pairwise percentage identities of both nucleotide and predicted amino acid sequences among12P. citri P450genes (11P450s obtained in this research and one P450gene CYP4CF1cloned in our previous study). The percentages of nucleotide sequence identities were36.47-96.39%and the pairwise percentages of their encoded proteins were16.29-96.39%. To analyze the phylogenetic relationships,75P450s of Tetranychus urticae were downloaded from BIOINFORMATICS&SYSTEM BIOLOGY and aligned to the12P. citri P450genes. A phylogenetic tree was constructed with MEGA5.05applying the Maximum Likelihood method and revealed that the12P. citri P450s belonged to3Clans (CYP2Clan, CYP3Clan and CYP4Clan). Additionally, CYPs within the same family were clustered in the same branch. The CPR and CYB5genes were also isolated from P. citri with the GenBank accession numbers of KJ173881and KJ173882. The PcCPR cDNA sequence contained an ORF of2,061nucleotides encoding686amino acids, while the PcCYB5cDNA sequence contained an ORF of324nucleotides encoding107amino acids. A multiple alignment of the amino acid sequences was conducted among PcCPR and other three arthropod CPRs. The result showed that PcCPR shared the greatest identity with T. urticae CPR and consisted of three conserved domains such as FMN, FAD and NADPH binding regions. Phylogenetic analysis suggested that CPRs from the same class were grouped together. PcCPR originated from a same evolutionary root with the CPR in T. urticae with the bootstrap value of100, which was consistent with the result of homology analysis. In addition, we generated the protein sequence alignment among CYB5s and found the typical conserved motif (the heme binding region) at the N terminus. Moreover, PcCYB5also had the highest homology with T. urticae CYB5.4Transcription profiles of P450and its relative genes from P. citri4.1Transcription profiles of P450genes4.1.1Developmental expression profiles of P450genesDevelopmental expression profiles of12P450genes containing entire coding region and14Unigenes were analyzed by RT-qPCR. The results showed that the transcripts of26P450genes were detected at all the life stages of P. citri. CYP4CF1, CYP4CL2, CYP392A24and CYP392B6were highly expressed at larval stage, while higher mRNA levels of CYP385C7and CYP389D1were observed at nymphal stage. Additionally,10P450Unigenes had the highest relative expression quantities in the larvae or nymphs. It was inferred that the16P450genes above might be involved in the detoxification process during larval and nymphal stages. The expression levels of CYP384A2and two Unigenes were increased during the development of P. citri, which indicated that the metabolic capacity also increased in ontogeny of the citrus red mite. We also found the expression levels of CYP392A22, CYP392A24, Unigene1146and Unigene27622were significant higher in the eggs, which suggested that these four P450genes might be associated with xenobiotic metabolism in the egg stage. The expression quantities of CYP385C8and CYP392F1were relatively stable, which suggested that the two genes might play important roles during the whole life of P. citri.4.1.2Response profiles of P450genes under xenobiotic stress To determine the response of P450genes, three acaricides (abamectin, fenpropathrin and pyridaben) and citral were chosen in this study. Based on the bioassay, the mites were treated in two different manners:time treatment (12,24and36h) and concentration treatment (LC10, LC30and LC50). The RT-qPCR analyses revealed that the transcription response of P450genes was very apparent after treatment with abamectin. Two CYP385members (CYP385C7and CYP385C8) was up-regulated more than25-folds and four CYP392genes (CYP392A22, CYP392A23, CYP392A25and CYP392B6) transcripts increased41.46,68.66,25.56and11.34-folds, respectively. For time course effect of abamectin on the expression patterns of P450s, the majority of the26genes had a highest mRNA level after24h and only two Unigenes transcripts reached the peak12h later. Compared to the P450genes above, CYP4CL2and CYP392F1exhibited a slower response. Two CYP385genes(CYP385C7and CYP385C8) expression levels were highest when treated with LQ10abamectin, the relative expression quantities of five CYP392members (CYP392A22, CYP392A23, CYP392A24, CYP392A25and CYP392F1) increased up to the maximum value after exposure to sub-lethal concentration (LC30) abamectin. Under the tress of abamectin at a median lethal concentration (LC50), the CYP4s(CYP4CF1and CYP4CL2) transcripts reached the highest level.Most of the detected P450genes could be induced by fenpropathrin, and the relative quantity of CYP4CL2was higher than other genes (4.48-folds). Following exposure to fenpropathrin for12h, four CYP392genes (CYP392A24, CYP392A25, CYP392B6and CYP392F1) as well as CYP384A2and CYP389D1reached the maximum expression. The highest transcription levels of two CYP4s were both observed24h later. After treatment with LC30fenpropathrin, the five genes CYP4CF1, CYP4CL2, CYP392A22, CYP392B6and CYP392F1were significantly up-regulated and the expression levels of them reached the peak. The results also showed that CYP385C8could be significant induced by the LC50of fenpropathrin. It could be indicated that these genes might participated in the detoxification process for fenpropathrin of P. citri.Unlike treatments with abamectin and fenpropathrin, the number of P450genes which were up-regulated reduced and the changes of relative quantities were less obvious after exposure to pyridaben. CYP385C7, CYP385C8and CYP392A22transcripts reached the peak after pyridaben treatment for12h, which suggested that the three CYPs might be involved in the detoxification of this acaricide during the early stage. The expression levels of CYP4CL2and four CYP392members (CYP392A23, CYP392A24, CYP392A25and CYP392B6) reached a maximum after24h or36h treatment and these genes might play roles in resisting the pyridaben stress during the middle and late stages. Only the mRNA level of CYP4CL2could be induced to the peak by LCio pyridaben concentration, while three CYP392genes and CYP385C8might be associated with the metabolism of high concentration pyridaben.The expression of all the12P450genes containing full-length cDNA increased significantly when treated by citral. The highest mRNA levels of most P450genes were detected after24h or36h treatment, which indicated that the response to citral of P450s in P. citri had a lag-effect. Moreover, qPCR analyses revealed that the high concentration of citral could lead to an obvious induction effect on P450s. There were ten P450genes containing entire ORF and eight Unigenes reached the highest expression level after exposure to citral at the concentration of LC50. The results also showed that the expression changes of CYP385C7, CYP385C8, CYP392A23, CYP392B6and CYP384A2were higher than other P450s (>8-folds increase), which could be inferred that the five CYPs might have important roles in the detoxification of citral and adaptation to the host environment.4.1.3Response profiles of P450genes induced by sodium barbiturateThe sodium barbiturate exhibited significant induction effects on the majority of P450genes in P. citri and the highest induction levels were detected after treatment for24h or36h in most cases. It was also found that a positive correlation existed between the relative expression quantities of five CYPs (CYP392A23, CYP392A25, CYP392B6, CYP384A2and Unigene9596) and the induction concentrations.4.2Transcription profiles of PcCPR and PcCYB5genes4.2.1Developmental expression profiles of PcCPR and PcCYB5genesThe expression levels of PcCPR at larval and nymphal stages were significantly higher than that in other two stages. The highest expression quantity was observed in larvae, while the mRNA level was the lowest in eggs. The developmental expression profile of PcCPR was consistent with the P450s activities during the life stages, indicating that PcCPR was mainly involved in the detoxification process mediated by P450s during larval and nymphal stages. Moreover, this phenomenon also suggested that a cooperative action existing between CPR and P450s in P. citri. The highest mRNA level of PcCYB5was detected in nymphs, which indicated that PcCYB5was likely to play an important role in delivering the electrons to P450catalysis.4.2.2Response profiles of PcCPR and PcCYB5genes under xenobiotic stressThe qPCR results showed that PcCPR exhibited a rapid response under the abamectin stress. The PcCPR transcripts reached a maximum after exposure to LC10abamectin for12h. The expression of PcCPR was inhibited by fenpropathrin at the concentrations LC10and LC50, while the sub-lethal concentration (LC30) could significantly induced PcCPR transcripts. Pyridaben showed the strongest induction effect on PcCPR and the citral treatments with different concentrations and times resulted in the up-regulation of PcCPR expression. Thus we concluded that PcCPR could be involved in the metabolic process for xenobiotics of P450. The expression of PcCYB5was significantly up-regulated after treatment with three acaricides and citral. It was also found that a complementary effect existed between the expression profiles of PcCYBS and PcCPR, which indicated that the cooperation of the two genes would ensure the high-speed transfer of electrons.4.2.3Response profiles of PcCPR and PcCYBS genes induced by sodium barbiturateThe qPCR analyses showed that both PcCPR and PcCYBS expression levels increased significantly after sodium barbiturate induction. These two genes transcripts rose with the increase of treatment time and concentration.5Eukaryotic expression of P450genes from P. citriIn order to study the function of CYP392A24and CYP392F1, we conducted the eukaryotic expression experiment with Bac-to-Bac baculovirus expression system. Two recombinant virus expression vectors Bacmid-CYP392A24and Bacmid-CYP392F1were successfully constructed in the use of double digestion of XhoⅠ/HindⅢ and BamHI/PstI restriction enzymes as well as the DNA recombination technology. Transfecting Sf9cells with the recombinant vectors, we finally obtained the expression products. The P450contents were determined by reduced CO-difference spectrum, but the CYP392A24and CYP392F1proteins were proved to be inactive. Therefore, the expression and protein purification system is still need to be improved in the future. However, the generation of eukaryotic expression system for P. citri P450s will provide the technical foundation for the functional verification of P450s and research on the detoxification capacity of target genes.In summary, we conducted the resistance monitoring in different populations of P. citri and compared the major detoxification enzymes activities. The transcriptome database of P. citri was generated via high throughput sequencing technology. Based on the transcriptome data, we identified the genes related to detoxification and analyzed their putative metabolic pathways. Using RT-PCR and RACE techniques,11P450genes as well as PcCPR and PcCYB5were isolated from P. citri. Transcription profiles of26P450genes (12CYPs containing entire coding region and14Unigenes), PcCPR and PcCYB5were determined through qPCR technique from P. citri including different developmental stages, acaricides and citral stress and sodium barbiturate induction. As a result, a series of P450genes involving in the detoxification of xenobiotics were identified. Furthermore, we generated the eukaryotic expression system for P. citri P450s and constructed two recombinant virus expression vectors. The results enriched the P450genes information of P. citri and will provide basic data for exploring the resistance related P450genes. Additionally, our study will promote the research on the metabolism resistance mechanism mediated by P450s. |
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