The CeRNA Mechanism Mediates Cyflumetofen Resistance In Tetranychus Cinnabarinus (Boisduval)

Tetranychus cinnabarinus is one of the most important agriculture mite pests.It is often difficult to manage because of its strong host adaptability and serious resistance to acaricides.As a new mitochondrial electron transport inhibitor,cyflumetofen shows striking activity on kinds of pest mites.Another advantage of this acaricide is its security to environment and the non-target organisms,so,it has a wide application prospect in the control of pest mites.Differential expressions of detoxification genes are one of the most important mechanisms involved in acaricide-resistance in mites.The regulation pathway of resistance genes expression is becoming a research focus in the field of resistance mechanism.The long non-coding RNA mediated competing endogenous RNA(ceRNA)offers a new opportunity to investigate post-transcriptional regulation of those genes.However,study on function of ceRNA in mite resistance,and even in physiological changes of arthropods are still unknown.In this work,we systematically investigated the function of the key GST gene in cyflumetofen-resistant T.cinnabarinus.Meanwhile,we comprehensively analysed the ceRNA regulation pathway,which mediated overexpression of GST,and its function in cyflumetofen resistance in T.cinnabarinus.The main results were as follows:1.Identification and functional analysis of cyflumetofen-resistant GST gene in T.cinnabarinus.The results of enzyme activity assay showed that the close relationship between GSTs activity and cyflumetofen-resistance,and the activity of GSTs increased along with the development of resistance.Compared with SS strain,6 of 13 GST genes were overexpressed in CyR strain(the upregulation was between 3.0-5.3 folds),in which the TcGSTm02 emerged the most sensitivity against the resistance change(its expression changed with significance from one resistant stage to the next),suggesting that the important role of TcGSTm02 in cyflumetofen-resistance.The catalytic activity of recombinant TcGSTm02 against CDNB was 6.82±0.27?mol/mg pro/min,which was32 times higher than the activity of crude GSTs in SS strain.The results of HPLC and LC-MS/MS assay indicated that recombinant TcGSTm02 could effectively decompose cyflumetofen,and catalyze GS~-to conjugate with cyflumetofen.2.Selection of miRNAs targeting GST gene and their regulatory relationshipBased on miRNA transcriptome data of T.cinnabarinus,the regulatory relationship between miR-133-5p and TcGSTm02 was predicted by RNAhybrid,MicroTar,and PITA.The expression level of miR-133-5p was significantly lower in CyR than SS strain,whereas TcGSTm02 expression was significantly higher in CyR than in SS strain.Overexpression or inhibition of miR-133-5p caused an opposite expression trend in TcGSTm02,which indicated that miR-133-5p negatively regulated the expression of TcGSTm02 in T.cinnabarinus.This relationship was further demonstrated by the dual luciferase report system,in which the relative luciferase activity of TcGSTm02 3'UTR was significant reduced compared with the negative control after transfecting miR-133-5p into the cell,demonstrating that miR-133-5p regulates the expression of TcGSTm02 by binding to its 3'UTR.3.Identification of lncRNAs and prediction of underlying lncRNA-miRNA-GST regulation pathway in T.cinnabarinusA total of 4,454 lncRNAs were identified in T.cinnabarinus by RNA-seq,further,lincRNA_Tc13743.2 and lincRNA_Tc1015.2 were predicted by RNAhybrid,RNA22,and PITA for having miR-133-5p binding sites.The results of RT-qPCR showed that the expression level of lincRNA_Tc13743.2 was significantly higher in CyR than SS strain,whereas there was no difference in lincRNA_Tc1015.2's expression between the two strains,suggesting that lincRNA_Tc13743.2 acted as the“sponge”of miR-133-5p.Expression-based subcellular localization assay showed that lincRNA_Tc13743.2,as well as TcGSTm02 and miR-133-5p were primarily expressed in cytoplasm of T.cinnabarinus cells.Therefore,the candidate regulatory pathway,which mediated overexpression of TcGSTm02,was lincRNA_Tc13743.2-miR-133-5p-TcGSTm02.4.Investigation of lncRNA-miRNA-GST regulation pathway by biotin-avidin and AGO1 immunoprecipitation assayBiotin-labeled miR-133-5p was overexpressed in T.cinnabarinus by feeding,followed by streptavidin-based pull-down assay.The results showed that,a significantly higher enrichment of lincRNA_Tc13743.2 and TcGSTm02 in the miR-133-5p-captured fraction compared with the negative-captured fraction,demonstrating that both lincRNA_Tc13743.2 and TcGSTm02 could bind to miR-133-5p.The results of AGO1 immunoprecipitation assay showed that lincRNA_Tc13743.2 and TcGSTm02 enrichment ratios were significantly higher in the miR-133-5p-fed than the negative control group,which indicated that both lincRNA_Tc13743.2 and TcGSTm02bind to miR-133-5p-associated silencing complex(RISC).5.Study on the sponge function of lncRNAThe co-localization signals of lincRNA_Tc13743.2 and miR-133-5p were found in mite's tissue,especially in the gut by fluorescence in situ hybridization.The results of dual luciferase report assay showed that miR-133-5p can bind to lincRNA_Tc13743.2and TcGSTm02,respectively.More importantly,overexpressed lincRNA_Tc13743.2and miR-133-5p in HEK293T cells significantly increased the relative luciferase activity of TcGSTm02,demonstrating that lincRNA_Tc13743.2 could act as a“sponge”for miR-133-5p,thereby reducing binding of miR-133-5p to TcGSTm02.6.Study on the function of lncRNA in cyflumetofen resistance by RNAiThe results of RNAi showed that in SS strain,silencing of lincRNA_Tc13743.2expression levels by 60%using a specific siRNA,led to a significant decrease in the expression level of TcGSTm02 by 77%,compared with the siGFP group.However,there was no difference in miR-133-5p expression level between the silenced and control group.Similar results were observed in CyR strain.These results indicated that lincRNA_Tc13743.2 promoted the expression of TcGSTm02 primarily by inhibiting miR-133-5p function.Data of bioassay showed that silencing lincRNA_Tc13743.2expression significantly increased the sensitivity against cyflumetofen in SS strain,as well as in CyR strain.Knocked down the expression of lincRNA_Tc13743.2 by 68%significantly decreased the resistance level of CyR strain by 28.7-fold,demonstrating that the important role of lincRNA_Tc13743.2-miR-133-5p-TcGSTm02 pathway in cyflumetofen resistance in T.cinnabarinus.In summary,this study systematically demonstrated that overexpressed lincRNA_Tc13743.2 competed for miR-133-5p binding and weakened the inhibitory effect of miR-133-5p on TcGSTm02 expression,thereby increasing TcGSTm02expression levels and cyflumetofen-resistance in T.cinnabarinus.Our results not only demonstrated the presence of a lncRNA-miRNA-mRNA pathway that regulates the pesticide/acaricide resistance in arthropod,but it also provided some theoretical basis for the reasonable use of cyflumetofen and the control of resistance in mites.