Mechanism Analysis Of UGT201D3 Associated With Abamectin Resistance In Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite,Tetranychus cinnabarinus?Boisduval?,is one of the major agricultural pests which is difficult to control.Up to now,its control has been mainly based on chemical control,which also led to the growing problem of its resistance.Abamectin is a macrocyclic lactones compound with superior activity of insecticide and miticidal nematocides,but with its large and unreasonable use,various pests?or mites?have been developed highly resistance to it.At present,studies on the mechanism of pests?or mites?resistance have found that the increase of the metabolic capacity of various detoxification enzymes including cytochrome P450,carboxylesterase,glutathione-S transferase and so on are important mechanisms of insect resistance.Uridine diphosphate glycosyltransferases?UDP-glycosyltransferases,UGTs?are widely distributed in the organisms and responsible for the glycosylation,which can catalyze the activation of UDP-sugar transfer the glucose to the receptor,thus regulating the biological properties,solubility,intercellular transport or other properties of the receptor.Many studies have shown that UGTs plays an important role in detoxification and metabolism in mammals,but there is a lack of research on the relationship between UGTs and resistance in insects?or mites?.In the results of the DNA microarray of T.cinnabarinus,a UDP-glycosyltransferases gene,Unigene9165_A,was found to be up-regulated about 3.82-fold in abamectin resistant strains of T.cinnabarinus.On this basis,this paper studied the relationship between UGTs and abamectin resistance in T.cinnabarinus.The main contents and results are as follows:1.The toxicity of susceptible strain?SS?and abamectin resistant?AbR?strain in T.cinnabarinus were assayed by the residual coated vial?RCV?method.After inhibiting the activity of UGTs in mites by UGT specific inhibitor 5-nitrouracil,the toxicity of abamectin in SS and AbR strains separately increased by 20.00%and 56.98%,which was shown that UGTs play a more prominent role in AbR strains,suggesting that they are involved in the development of avermectin resistance in T.cinnabarinus.2.Activity of UGTs enzymes in SS and AbR strains of T.cinnabarinus were detected.The results showed that the UGTs activities of SS and AbR strains were0.74±0.04 nmol/mg.pro.min^-1 and 1.73±0.29 nmol/mg.pro.min^-1,respectively.The activity of UGTs in AbR strain was significantly higher than that in SS strain.Therefore,it is speculated that the increase of UGTs activity was probably correlated with T.cinnabarinus's resistance to abamectin.3.The UGT gene Unigene9165_A,which was up-regulated in the abamectin resistant strain of T.cinnabarinus,was cloned and analyzed its bioinformatics.The full-length cDNA of this gene is 1383bp and encodes 460 amino acids.Its GeneBank accession number was KX905079.The predicted protein molecular weight was53.49kDa and the theoretical isoelectric point was 5.74.The gene was named UGT201D3 by the UGT Nomenclature Committee.Bioinformatics analysis showed that the gene has the structural domain of UDPGT gene family and belongs to the GT-B superfamily,containing the characteristic sequence of the UGT gene family.Phylogenetic analysis showed that the gene belongs to the UGT201 family and has high homology with UGT201D2 gene of two-spotted spider mites.4.The mRNA expression patterns of UGT201D3 gene in different strains of T.cinnabarinus,different developmental stages and drug stress were analyzed by qPCR.The results showed that the UGT201D3 had the lowest expression level in eggs,followed by nymphs,adults and larvae.Compared to SS strain,the expression of the gene was significantly higher in AbR strain,which is consistent with the result of the DNA microarray.After low dose abamectin induction 6h,12h and 24h,the expression of UGT201D3 in SS was not significantly different from that of the control.While in AbR strain,the expression of UGT201D3 increased significantly after 6h and 12h of abamectin stress,which were 2.63-and 2.08-fold of the control respectively,but no significant difference after 24h.5.The RNAi of UGT201D3 was studied using the"leaf dish absorption feeding method".After feeding UGT201D3-dsRNA,the silencing efficiency of SS was 48.82%and UGTs enzyme activity was reduced by 39.30%.The sensitivities to abamectin before and after treatment were no significant differences under the LC₃₀ and LC₅₀0 dose,while the sensitivity under the LC₇₀ dose was increased by 7.79%.In AbR strains,the transcript levels of UGT201D3 was decreased by 51.25%and the enzyme activity of UGTs decreased 63.20%.The mortality of LC₃₀,LC₅₀ and LC₇₀0 of abamectin are increased significantly by 16.42%,11.94%and 22.99%,respectively.6.The prokaryotic expression vectors?UGT201D3-pCold?/pET-28a and UGT201D3q-pCold?/pET-28a?were constructed and expressed active soluble protein.The measured K_m and V_max values of the recombinant protein were 22.73±5.87?M and11.08±2.77nmol/mg.pro.min^-1,respectively.The half maximal inhibitory concentration(IC₅₀)of abamectin was 57.50±3.54?M and the inhibition constant Ki was 9.9±6.2?M.The drug decomposition experiments showed that the recombinant protein could catabolize abamectin.The catabolism rates at 150?g/mL and 300?g/mL protein concentrations were 15.77%±3.72%and 16.75%±1.34%,respectively.