Cytochrome P450 And Carboxylesterase Mediated Insecticide Resistance In Rhopalosiphum Padi

The bird cherry-oat aphid,Rhopalosiphum padi（L.）（Hemiptera:Aphididae）,is an important pest of wheat worldwide.It damages wheats by direct feeding and transmission of the barley yellow dwarf virus（BYDV）,which causes an economically important disease in small grains,leading to reduced quality and yield of wheat every year.Chemical insecticides spraying is the major method to control this pest.Unfortunately,the extensive application of insecticides has resulted in the insecticide resistance in field populations of R.padi.Previous studies suggest that the cytochrome P450 monooxygenases（P450s）system and carboxylesterases（CarEs）are involved in the detoxification of xenobiotics,including insecticides in insects.To our knowledge,the molecular mechanism of P450 or CarE-mediated insecticide resistance in R.padi is still unavailable.In the present study,P450 genes were characterized based on the R.padi genome data published in public database.Quantitative PCR was used to analyses the expression levels of the P450 genes between the isoprocarb resistant and susceptible strains.RNA interference of the relative P450 gene was used to investigate the function of the gene in the resistance of the aphid to carbamate insecticides.A new P450 CYP6CY3-3 was cloned from R.padi.The transcripts of CYP6CY3-3 gene were analyzed between imidacloprid-resistant and susceptible strains.A preliminary study of the regulation mechanism of CYP6CY3-3 gene expression in the transcriptional level was conducted.Using the degenerate primers and RACE-PCR,the full-length cDNA coding for NADPH-cytochrome P450 reductase（CPR）was cloned and identified.In order to analyze the relationship between RpCPR and insecticide resistance,the mRNA of RpCPR gene in isoprocarb-resistant and imidacloprid-resistant strains were compared with that in the susceptible strain.Furthermore,polymorphisms of RpCPR were detected in the individuals of the field populations.Multiple analyses were conducted to reveal the function of carboxylesterase in the resistance to carbamates and pyrethroids in R.padi.Our studies will contribute to understanding the molecular mechanism of insecticide resistance in R.padi,and provide knowledge for the development of integrated pest management strategies for control of the pest.The main findings are as follows:1.The identification,cloning of P450 genes and the role of P450 in the isoprocarb resistance of R.padiBased on the genomic data of R.padi,the initial P450 gene sequences was obtained according to the sequence annotation and P450 conserved structural analyses.Through sequence multiple comparison and secondary annotation with NCBI,a total of 54 P450genes were identified.These P450 genes are distributed in four different clades,including nine families and 17 subfamilies.CYP2,CYP3,CYP4 and mitochondrial clades consist of nine,24,15 and six P450 genes,respectively.Fifty of the 54 P450 genes were successfully amplified in the R.padi by RT-PCR.Rpa00969 and Rpa18866 were not obtained by PCR amplification with multiple pairs of primers.Rpa00968,Rpa16802 and Rpa16803 were amplified and nucleotide showed those were fragments of the same P450 gene.Bioassays showed that the isoprocarb resistance strain（IS-R）of R.pad developed an approximate 35-fold resistance to isoprocarb compared with the susceptible strain（SS）.The cross-resistance of the R.padi IS-R strain to three carbamate insecticides methomyl,carbofuran and indoxacarb was measured,and the results showed that IS-R strain had cross-resistance to these three insecticides with resistance ratios of 12.56-,8.74-and6.07-fold,respectively.The enzyme activity assay showed that the P450 enzyme activity in the resistant strain was 1.78-fold higher than that in the susceptible strain,indicating that P450 plays an important role in the isoprocarb resistance of R.padi.The mRNA levels of CYP3 and CYP4 P450 genes in isoprocarb resistant and susceptible strains were detected by qPCR.The results indicated that the expressions of CYP6CY7,CYP6CZ1 and CYP4CJ1 were significantly up-regulated in the IS-R compared with SS.The changes of the above three P450 genes expression were further investigated in response to isoprocarb treatment,and the result showed that the CYP4CJ1 gene was the only P450 gene significantly up-regulated in mRNA level in different aphid strains and multiple isoprocarb concentrations.A RNAi via injection was performed to clarify the role of CYP4CJ1 on the susceptibility to isoprocarb in R.padi.Twenty-four hours post-injection of dsCYP4CJ1,the mortality of IS-R strains significantly increased 34.12%,23.05%,26.87%and 30.58%under LC₅₀ concentrations of isoprocarb,methomyl,carbofuran and indoxacarb,respectively,in comparison with the injection of ds GFP.These results indicated that CYP4CJ1 gene was involved in resistance to carbamate insecticides in R.padi.2.The cloning and transcriptional regulation mechanism of CYP6CY3-3 gene in R.padiBased on the previous CYP6CY3 genes research in R.padi,a new CYP6CY3 gene,CYP6CY3-3,was cloned.Amino acid sequence analysis demonstrated that CYP6CY3-3constituted the CYP6CY3 gene cluster together with the previously reported CYP6CY3-1 and CYP6CY3-2.The qPCR result showed that the CYP6CY3-3 gene was significantly up-regulated at the transcriptional level in the imidacloprid resistant strain（IM-R）.In order to analyze the regulation mechanism of CYP6CY3-3 expression in the transcriptional level,the 5’-flanking region of CYP6CY3-3 gene was cloned by genome walking technique in IM-R and SS strains.By analyzing 5’-flanking sequences from individuals of both resistant and susceptible strains,nucleotide diversity of 5’-flanking sequences of CYP6CY3-3 gene in IM-R was significantly lower than that in SS.The prediction of transcription factor（TF）binding boxes revealed the presence of multiple functional elements in the 5’-flanking region of CYP6CY3-3 gene.The function of5’-flanking region was tested by luminescent reporter assay in Sf9 cell line.The TF binding boxes in the-1044^-757 fragment made significant contribution to up-regulation.3.MolecularCloning,expressionpatternandpolymorphismof NADPH-cytochrome P450 reductase in R.padiNADPH-cytochrome P450 reductase（CPR）functions as an electron transporter（redox partner）,accepting electrons from NADPH and transferring them to P450s,and affect the activities of P450s.Only one CPR gene exists in the genome of insect.A full-length cDNA of RpCPR encoding 681 amino acids was cloned from R.padi.The amino acid sequence analysis showed that RpCPR exhibited conserved regions such as FMN,FAD and NADPH binding regions,and shared the greatest identify with to the CPR gene of Acyrthosiphon pisum.The expression of RpCPR gene was specificity in different developmental stages of R.padi,with the highest expression level found in the second instar and the lowest in adult.Expression levels of RpCPR in isoprocarb-resistant and imidacloprid-resistant strains were3.74-and 3.53-fold higher,respectively,than that in the susceptible strain.Apterous adult aphids of the SS strain were exposed to the LC₃₀ concentrations of isoprocarb and imidacloprid.RpCPR expression increased 1.66-to 2.18-fold after exposure to the isoprocarb.The RpCPR mRNA level was significantly increased at 3 h（1.85-fold）and 6 h（2.08-fold）after imidacloprid treatment,and then recovered to a normal level at 12 h（1.16-fold）and 24 h（1.19-fold）.The results indicated that RpCPR was associated with resistance to imidacloprid and isoprocarb.The coding region of RpCPR in 167 individuals from 11 geographic populations were analyzed.A total of 146 RpCPR haplotypes were identified by 334 polymorphic sites,65 of which were found in two or more individuals.The A627T,G1362A,C1450T and A1536TA polymorphic sites were identified in 31.7%、35.3%、35.3%and 30.5%individuals,respectively.Totally,194 missense mutation sites were defined in the amino acid sequence of RpCPR,of which,31 missense mutations belonged to parsimony-informative sites.The Pro484Ser mutation site was found in 59 individuals from all 11 geographic populations.4.Functional analysis of a carboxylesterase gene associated with isoprocarb and cyhalothrin resistance in R.padiCarboxylesterase activities of different strains were determined usingα-NA as a substrate.Results showed that isoprocarb resistant and cyhalothrin resistant strains exhibited significantly higher carboxylesterase activity（2.20-and 2.05-fold）compared with the susceptible strain,respectively,which suggest that carboxylesterase plays an important role in the insecticide resistance of R.padi to isoprocarb and cyhalothrin.Seven putative carboxylesterase genes were obtained from the transcriptome data of R.padi.Relative expression analysis showed that the CL2102 were 4.99-and 2.73-fold higher in IS-R and CY-R（cyhalothrin resistant strain）than that in SS,respectively.We then focus on the analysis of CL2012 gene.The full-length cDNA of CL2012 sequence was cloned and named as RpCarE.Homology analysis of RpCarE against the already published carboxylesterases indicated that carboxylesterase shared high identity with Myzus persicae FE4-like esterase,A.pisum FE4 esterase,Aphis glycines carboxylesterase and A.gossypii carboxylesterase.The alignment analysis shows that RpCarE has highly conserved residues that form a catalytic triad（S185-H432-E312）.To express RpCarE in vitro,the coding region was inserted into the pET-28a expression vector and expressed in the BL-21（DE-3）Escherichia coli strain.The purified RpCarE protein exhibited catalytic efficiency toward the model substrateα-NA with a Kcat/Km of0.13μM^-1·s^-1.The catalytic activity of RpCarE recombinant protein toward isoprocarb and cypermethrin was measured by HPLC analysis respectively.The results showed that the fusion protein had significant activities toward both isoprocarb and cyhalothrin.A molecular docking simulation was conducted using the homology mode of RpCarE with isoprocarb and cyhalothrin by SYBYL X2.0 software.The modelling and docking analyses showed that isoprocarb and cyhalothrin fit snugly into the catalytic pocket of RpCarE,positioning the molecule close to catalytic triads（Ser185-His432-Glu312）.These findings suggest that RpCarE plays an important role in metabolic resistance to carbamates and pyrethroids in R.padi.