Study On Cyflumetofen Resistance Regulated By Tci-miR-1-3p Targeting TCGSTM4 In Tetranychus Cinnabarinus

Tetranychus cinnabarinus is a serious agricultural pest mite,which feeds on more than one hundred agricultural crops,due to their living habit and the massive abuse of chemical insecticides,T.cinnabarinus has developed resistance to acaricides rapidly.Cyflumetofen is a novel benzoyl acetonitrile acaricide,was first registered in Japan in2007,it since has been used in 15 countries to control mites in agricultural crops,and there were many reports about mites'resistance to cyflumetofen.MicroRNAs?miRNAs?are important factor that negatively regulate the stability and translation of mRNA transcripts,in recent years,cases of miRNAs involved in insecticide resistance through targeting insecticide resistance related genes in arthropods have been documented.MiR-1 is a well studied conservative and abundant miRNA involved in growth development and immune responses among insects,so far,little is known about its role on pesticide-resistance in arthropods.In previous study,we explored the cyflumetofen resistance related target gene of miR-1 in T.cinnabarinus?Tci-miR-1-3p?,moreover,the function of the target gene in the formation of the cyflumetofen resistance was explored,these studies formulated the role of Tci-miR-1-3p in the formation of cyflumetofen resistance in T.cinnabarinus.The main results of experiments are as following:1.Characterizing Tci-miR-1-3p expression in susceptive and resistant strainQuantitative real-time PCR was performed to detect the expression level of Tci-miR-1-3p in cyflumetofen-sensitive strain?SS?and cyflumetofen resistant strain?CYR?,the expression level of Tci-miR-1-3p in CYR was significantly down-regulated compared to that in SS.The result indicated an involvement of Tci-miR-1-3p in cyflumetofen-resistance in T.cinnabarinus.2.Screening and verifying the targets of Tci-miR-1-3pIn order to further explore the cyflumetofen resistance mechanism mediated by Tci-miR-1-3p,6 biological softwares?miRanda and RNAhybrid,and so on?were used to predict the targets of Tci-miR-1-3p,19 genes?glutathione S-transferase?TCGSTM4??EF-Hand Protein?EFHAND2??cytochrome P450?CYP307A1?and carboxylesterase?TCCarE 14?and so on?were selected as potential targets of Tci-miR-1-3p.Then feeding method was used to verify the targeting relationship between Tci-miR-1-3p and its potential targets,and the results showed that the expression level of Tci-miR-1-3p was significant higher in feeding Tci-miR-1-3p mimic treatment than those in feeding DEPC water and negative control mimic treatments,the expression of TCGSTM4 and EF-HAND2 were significantly down-regulated to 0.57-and 0.55-fold of the control after increasing the content of Tci-miR-1-3p in T.cinnabarinus.The results indicated there might be a negative regulation between Tci-miR-1-3p and TCGSTM4 and EFHAND2.According to the reports about the function of these two genes,TCGSTM4,which may be invoved in the insecticide resistance in mites,was slected as the target for further target verification.Dual-luciferase reporter gene assay was conducted to verify the regulation relationship between Tci-miR-1-3p and TCGSTM4:Compared with the negative control(NC control:co-transfection of negative mimic with the dual-luciferase reporter with the original 3'UTR of TCGSTM4?pmirGLO-UTR?into Human Embryonic Kidney 293T?HEK293T?cells,dual-luciferase assay showed that the firefly luciferase activity?with normalization to Renilla?decreased significantly to about 50%after co-transfection of Tci-miR-1-3p mimic with pmirGLO-UTR into HEK293T cells.However,co-transfection of Tci-miR-1-3p mimic with pmirGLO-MUTR,which has a mutated 3'UTR sequence of TCGSTM4,did not reduce luciferase activity.These results indicated that Tci-miR-1-3p could regulate TCGSTM4 expression by binding and acting on the 3'UTR sequence of TCGSTM4,in other words,the regulation relationship between Tci-miR-1-3p and TCGSTM4 was confirmed.3.The impacts of Tci-miR-1-3p on TCGSTM4's expression and T.cinnabarinus's tolerance against cyflumetofenFurthermore,oversupply or inhibition of Tci-miR-1-3p by oral feeding mites with Tci-miR-1-3p mimic and Tci-miR-1-3p inhibitor were used to explore the influence of Tci-miR-1-3p on TCGSTM4's expression and mites'tolerance against cyflumetofen.After feeding Tci-miR-1-3p mimic,compared with controls,the transcription of TCGSTM4 was significantly decreased to 0.57-or 0.72-fold when the content of Tci-miR-1-3p increased to 1.51-fold or 2.93-fold in SS and CYR,respectively.Consequently,the protein expression level of TCGSTM4 was reduced dramatically to 0.47-and 0.61-fold in SS and CYR.On the other hand,compared with the control,reduced the content of Tci-miR-1-3p to 0.69-and 0.52-fold in SS and CYR strains led to increase gene expression of TCGSTM4to 1.29-and 1.32-fold at mRNA level and to 1.96-and 2.11-fold at protein level,respectively.Moreover,The subsequent bioassay data showed that the mortalities increased 14.01%and 14.86%in miRNA mimic-fed SS mites when treated with two cyflumetofen concentrations(LC₁₀ and LC₃₀),and those in CYR mites increased 16.69%and 20.07%,respectively;and the data showed that the mortalities decreased 12.12%and12.15%in miRNA inhibitor-fed SS mites when treated with two cyflumetofen concentrations(LC₃₀ and LC₅₀),and those in CYR mites decreased 14.37%and 24.13%,respectively.The negative regulation relationship between Tci-miR-1-3p and TCGSTM4was verified by oversupply or inhibition of Tci-miR-1-3p by oral feeding mites with Tci-miR-1-3p mimic and Tci-miR-1-3p inhibitor,and Tci-miR-1-3p exerted influence on the tolerance of T.cinnabarinus against cyflumetofen.4.Studies of the function of TCGSTM4 by RNA interference and responsive pesticide-stimulationTo further clarify the involvement of TCGSTM4 in the formation of cyflumetofen resistance in T.cinnabarinus,RNA interference?RNAi?was used to explore the function of TCGSTM4.The results showed that the transcription of TCGSTM4 reduced to 0.41-fold in the SS and 0.49-fold in the CYR compared with controls,the subsequent cyflumetofen-activity assay showed that the mortalities increased 11.69%and 15.69%in TCGSTM4 dsRNA-fed SS mites when treated with two cyflumetofen concentrations(LC₁₀ and LC₃₀),and those in CYR mites increased 16.72%and 19.19%,respectively;Mowever,a sublethal dose(LC₁₀)was adopted to stimulate the mites from SS and CYR,the expression of TCGSTM4 was significantly increased when the mites from CYR were exposed for 12 h and 24 h,but there was no significant changes in SS.The result of RNAi and responsive pesticide-stimulation further indicated the involvement of TCGSTM4 in the formation of cyflumetofen resistance in T.cinnabarinus.5.Studies of the function of TCGSTM4 by heterologous expression and pesticide metabolismHeterologous expression was adopted to obtain the recombinant TCGSTM4 protein with E.coli expression system.The SDS-PAGE analysis results showed that the molecular weight of the soluble protein was 26 kDa.Our results also showed that the specific activity of the recombinant TCGSTM4 for 1-chloro-2,4-dinitrobenzeen?CDNB,a specific substrate of glutathione S-transferase?was 428.90±12.98 nmol/mg pro.min^-1.And the CDNB conjugating activity of TCGSTM4 could be inhibited by cyflumetofen and de-esterified form of cyflumetofen and the true active molecule inhibiting the target in mites?AB-1?,the half maximal inhibitory concentration(IC₅₀)of cyflumetofen and AB-1 on the inhibition of CDNB conjugating activity of TCGSTM4 was determined to be 0.83±0.08 mM and 0.18±0.05 mM.Moreover,the results of decomposing assays showed that cyflumetofen could be degraded directly by the recombinant TCGSTM4protein,and the decomposing effect could be greater with more protein.The decomposing rate of cyflumetofen were 32.21%±2.44%,36.00%±5.83%and 41.79%±1.69%when treated with 20,40 and 60?g/mL of recombinant TCGSTM4,respectively.The results of heterologous expression indicated that the recombinant TCGSTM4 protein was glutathione S-transferase,and the interaction between TCGSTM4 and pesticide provide first direct evidence that the recombinant TCGSTM4 protein can significantly decompose cyflumetofen.