Abstract: 【Objective】 Tetranychus cinnabarinus is an important pest mite in agriculture. Resistance of this pest mite to various pesticides has developed quickly because of long-term chemical control, thus, it is of great significance to clarify the detoxification mechanism of T. cinnabarinus in order to carry out effective resistance management. Glutathione S-transferase (GST) is one of the major detoxification enzymes in Arthropods. In previous research, a highly expressed GST gene TcGSTM7 from T. cinnabarinus was identified, and the expression of this gene is inducible by pesticide. In this case, the objective of this study is to construct the recombinant protein of TcGSTM7 with prokaryotic expression system, analyze interaction between recombinant protein and pesticides (fenpropathrin and cyflumetofen), and test the pesticide susceptibility change of T. cinnabarinus after decreasing the expression of TcGSTM7 through RNAi.【Method】The sequence characteristics of TcGSTM7proteinwere annotated by BLAST in NCBI database. The structural domain, binding site, and active site were presented in a three-dimensional structure model, which was predicted by SWISS MODEL. TcGSTM7 was expressed by prokaryotic expression in Escherichia coli. GST activity of recombinant protein was tested with specific substrate, and its interaction with fenpropathrin and cyflumetofen was analyzed through activity inhibition test. Then, specific dsRNA was synthetized according to the sequence information of TcGSTM7. T. cinnabarinus was feed on the dsRNA through leaf disc method. Gene expression change of TcGSTM7 was detected by qPCR, then, susceptibility change of T. cinnabarinus to fenpropathrin and cyflumetofen was analyzed by bioassay. 【Result】Amino acid sequence analysis ofTcGSTM7 showed that the N-terminal domain contained a thioredoxin-like domain, which is a specific feature of Mu class GST. In addition, GSH binding site (G-site) was also located in the N-terminal domain. The C-terminal domain contained an alpha helical domain, and the substrate binding pocket (H-site) was included. This protein consisted of nine α-helices and four β-strands and contained the typical βαβαββα motif of the thioredoxin fold. Based on the three-dimensional structure prediction, recombinant protein of TcGSTM7 was constructed through prokaryotic expression. Molecular weight of this protein was 26 kD, which was the same as prediction. Its GST specific activity was 673.26 nmol·min^-1·mg^-1, and kinetic parameters were calculated as 0.71 mmol·L^-1 for K_m and 109.54 nmol·min^-1·mg^-1 for V_max. Analysis of interaction between pesticides and the recombined protein showed that both fenpropathrin and cyflumetofen could inhibit the activity of TcGSTM7. IC₅₀ was 0.038 mmol·L^-1 for fenpropathrin, and 0.2 mmol·L^-1 for cyflumetofen. Then, dsRNA was feed to T. cinnabarinus by leaf disc feeding method. qPCR data showed the expression of TcGSTM7 was significantly decreased by 52.88%, and bioassay was carried out to test susceptibility of the mites to these two pesticides. Results showed that susceptibility of T. cinnabarinus to fenpropathrin significantly increased by 9.0% and 12.3% at LC₃₀ and LC₅₀, and to cyflumetofen, it also significantly increased by 12.9% and 11.0% at LC₃₀ and LC₅₀ (P＜0.05).【Conclusion】The expression of TcGSTM7 could affect the susceptibility of T. cinnabarinus to fenpropathrin and cyflumetofen, and the interaction between recombinant protein of TcGSTM7 and these two pesticides was identified. These data indicate that TcGSTM7 is important for T. cinnabarinus during its metabolism process of fenpropathrin and cyflumetofen.

Key words: Tetranychus cinnabarinus, glutathione S-transferase (GST), RNAi, detoxification