| The carmine spider mite,Tetranychus cinnabarinus（Boisduval）,is an important agricultural pest that is widely distributed,harmful and difficult to control.Up to now,chemical pesticide is the most effective method to control mites,while T.cinnabarinus has developed resistance to various acaricides.As a novel benzoyl acetonitrile acaricide and semi-esterase precursor dependent acaricide,Cyflumetofen has been widely used in the field to control pest mites.At present,the resistance of cyflumetofen is mainly caused by the enhancement of detoxification enzyme activity,while transcription factors including nuclear receptors participate in the detoxification metabolism process of insecticide through transcriptional regulation of target genes in T.cinnabarinus.HR96 is a member of the nuclear receptor superfamily,and previous studies have showed that HR96 is a criticl factor for xenobiotic resistance.Based on the previous transcriptome analysis of T.cinnabarinus and the genomic database of Tetranychus urticae,this study focused on the function of TcHR96 in cyflumetofen resistant mechanism of T.cinnabarinus.The results are as follows:1.The gene sequence encoding nuclear receptor HR96 of T.cinnabarinus was cloned.The open reading frame of this gene is 1506 bp and encodes 501 amino acids.The predicted protein molecular weight was 56.88 k Da and the theoretical isoelectric point was 6.55.The gene was named TcHR96.Bioinformatics analysis showed that TcHR96 has typical LBD and DBD domains of nuclear receptor family and belongs to the NR1J superfamily,and it was found to be homologous to Tetur36g00260 in T.urticae.2.The m RNA expression pattern of TcHR96 in different strains of T.cinnabarinus and under exposure to acaricides were analyzed by q PCR,the results showed that compared to SS,the expression of TcHR96 was not significantly different in Fe R strain,but significantly higher in Cy R strain（18.2 folds）.After induction by cyflumetofen at LC10 and LC25 for,6h,12h and 24h,the expression of TcHR96 was not significantly different from that of the control,neither in SS or in Cy R.3.The RNAi of TcHR96 was carried out using the"leaf dish feeding method".After feeding TcHR96-ds RNA,the silencing efficiency of SS was 59%,and LC50 of cyflumetofen was significantly decreased from 2.9mg/L to 1.5mg/L;in Cy R,the silencing efficiency was 80%,and LC50 of cyflumetofen was significantly decreased from 253.3mg/L to 68.4mg/L.After TcHR96-RNAi,the cyflumetofen resistance ratio of Cy R versus SS decreased from 87-fold to 43-fold,indicating that TcHR96 is essential for cyflumetofen resistance in T.cinnabarinus.4.The prokaryotic expression vectors（p Cold II-TcHR96、p ET 28a-TcHR96、p ET30a-TcHR96和p Cold SUMO-TcHR96）were constructed and the recombinant protein was found in inclusion body.Then,the renaturation of p Cold II-TcHR96 inclusion body was applied according to dilution renaturation method,and finally,the soluble protein of TcHR96 was successfully obtained.5.DARTS method with fenpropathrin as control was used to detect the binding ability of recombinant protein TcHR96 to cyflumetofen and AB-1（the active metabolite of cyflumetofen）.DARTS results showed that cyflumetofen and AB-1 had obvious protective bands,proving that TcHR96 could bind to cyflumetofen and AB-1.The Kd value for TcHR96 and cyflumetofen was 234μM,for TcHR96 and AB-1 was130μM by Microscale Thermophoresis（MST）.6.Combining RNAi and transcriptome sequencing technology,three downstream detoxifying enzyme genes which regulated by TcHR96 potentially were obtained:TcGSTd08,TcGSTm02,and TcGSTm09.The promoter sequence of TcGSTm02 gene was cloned,and two potential nuclear receptor transcription factor binding sites were successfully predicted.The luciferase reporter assays were further conducted to determine the regulation of TcHR96 on TcGSTm02 promoters.The results showed that after co-transfection of the TcHR96 and TcGSTm02 gene promoters,the fluorescence activity was significantly higher than that of control（2.87 times）,and the regulatory effect of TcHR96 on TcGSTm02 was initially clarified. |
