| Bactrocera dorsalis is an important agricultural pest and invasive species in China.Due to global warming and the frequent transportation of fruits and vegetables,B.dorsalis has developed varying degrees of resistance to a variety of insecticides.It has been shown that the overexpression of detoxification metabolic enzymes is one of the reasons for insects developing metabolic resistance to multiple insecticides.UDPglycosyltransferase(UGTs)is an important secondary metabolic enzyme in insect metabolic resistance.However,the function and transcriptional regulation mechanism of UGTs in insecticide resistance of B.dorsalis remains unclear.Therefore,in this dissertation,resistance strains of B.dorsalis obtained through continuous screening using malathion in the laboratory were used to determine whether UGTs are involved in the resistance of B.dorsalis to malathion via biochemical toxicological analysis.The UGT genes were screened and identified at the genome-wide level,followed by family classification,phylogenetic analysis,and chromosome distribution to identify key overexpressed UGTs in malathion resistance strain(MR).Immunohistochemistry,RNAi,3D-structure simulation and molecular docking,heterologous expression and HPLC were used to determine whether important UGTs genes mediate malathion resistance of B.dorsalis.Comprehensive bioinformatics prediction and differential expression analysis of transcription factors in MR and MS strains,combined with RNAi,dual luciferase reporter system,ROS activator and inhibitor treatment,have revealed the molecular mechanism of ROS activation regulating the overexpression of important UGT genes in MR through the transcription factor pathway.The key findings were as follows:1 Biochemical and toxicological analysis of UGTs-mediated resistance to Malathion in B.dorsalisThe bioassay was carried out on MR and MS strains without exposure to malathion selected in laboratory by drip method.The results showed that the LD50 of MR and MS strains were 5,915.84 ng/ fly and 95.09 ng/ fly,respectively.The resistance ratio was62.21-fold.After treatment with UGT-specific inhibitors Sul and 5-Nul,the LD50 of the MR strain was reduced to 1,778.42 ng/ fly and 1,453.79 ng/ fly,respectively.The synergid ratios were 3.33 and 4.07,respectively.However,after the treatment with Sul and 5-Nul,the LD50 of the MS strain was 91.53 ng/ fly and 56.74 ng/ fly,respectively.The synergistic ratio was 1.04 and 1.68,respectively.These results showed that UGTs specific inhibitors had synergistic effect on the MR strain,but not on the MS strain.Meanwhile,the specific activity of UGTs in the MR strain was 3.72-fold higher than that in the MS strain,which indicated that the increase of UGTs enzyme activity played an important role in malathion resistance.2 Identification of the different UGT genes between MR and MS strains of B.dorsalisA total of 31 UGT genes were identified based on the B.dorsalis genome database,of which 30 genes had complete ORF.All the UGT genes contained two sugar receptor binding domains,and 29 UGT proteins contained four conserved domains and signal peptide.Phylogenetic analysis showed that BdUGT301,BdUGT302,BdUGT35,BdUGT36,BdUGT37,BdUGT49,and BdUGT50 gene families were closely related to Drosophila UGT genes.Six genes of the BdUGT429 family of B.dorsalis clustered into one branch,this maybe coursed by gene amplification.Except for BdUGT49D3,the other 30 UGT genes were located on four chromosomes.BdUGT301,BdUGT302,BdUGT35 and BdUGT429 gene families were closely distributed,close to each other and encoded in the same direction on the same chromosome.RT-qPCR was used to analyze the expression patterns of UGT genes in the 4-dayold adults of the MR and MS strains of B.dorsalis.The results showed that BdUGT301D2,BdUGT35G1,BdUGT429A1,BdUGT429A3,BdUGT429B4,BdUGT442A1 and BdUGT50B5 were significantly up-regulated in the MR strain.It is suggested that these genes played an important role in the resistance to malathion of B.dorsalis.Furthermore,BdUGT301D2 and BdUGT429A1 were overexpressed in the MR strain throughout the adult stage,suggesting that these two UGTs take an important part in the resistance of B.dorsalis to malathion metabolism.3 Functional analysis of BdUGT301D2 and BdUGT429A1 in mediating malathion resistance of B.dorsalisUsing RT-qPCR and immunohistochemistry,BdUGT301D2 and BdUGT429A1 were found to be abundant in the cell membrane and organelle membrane of the midgut and malpighian tubule cells of the MR strain,suggesting that B.dorsalis UGTs may participate in transmembrane transport of toxic substances for detoxification of malathion.After silencing BdUGT301D2 and BdUGT429A1 tested by RNAi and Western Blot analysis,the silencing efficiency was 69.68% and 57.55%,respectively,accompanied by a significant decrease in the corresponding proteins.Silencing BdUGT301D2 and BdUGT429A1 resulted in significantly higher malathion susceptibility of 23.19% and 20.42%,respectively.Subsequently,3D simulation and molecular docking analysis showed that both BdUGT301D2 and BdUGT429A1 have active sites for binding to malathion isomers,which may directly bind to malathion.The content of malathion was significantly reduced by 47.54% and 20.40% after coincubation with the recombinant protein obtained based from Sf9,indicating that the recombinant protein could directly degrade malathion.Further cytotoxicity assay found that the tolerance of Sf9 cells expressing the two UGT genes to malathion was significantly increased by 31.58% and 31.13%,respectively,indicating that BdUGT301D2 and BdUGT429A1 contributed to malathion detoxification of B.dorsalis.In conclusion,BdUGT301D2 and BdUGT429A1 may directly degrade or bind to malathion through glycosidation,thereby enhancing malathion resistance of B.dorsalis.4 Analysis of the regulation of BdUGT301D2 and BdUGT429A1 expression by transcription factors BdCncC/BdMaf-KThe promoter sequences of BdUGT301D2 and BdUGT429A1 were not different between MR and MS strains,suggesting that their up-regulation expression may be mediated by transcription factors.Based on bioinformatics prediction analysis,it was found that there were transcription factor binding sites of BdUGT301D2 and BdUGT429A1 both potentially bind to BdMaf-K.Among the 16 transcription factors that have been reported to be involved in the regulation of resistance,BdMaf-K was highly expressed 1.82-fold in MR strain compared with MS strain of B.dorsalis.Therefore,it is preliminarily speculated that BdMaf-K regulates the overexpression of BdUGT301D2 and BdUGT429A1.Given the importance of the CncC/Maf pathway in the regulation of insect resistance,bioinformatics analysis of BdCncC/BdMaf-K revealed that its functional domain was conserved and phylogenetically close to other insect CncC/Maf in Dipteran.Furthermore,the dual-luciferase reporter system revealed different regulatory mechanisms between BdUGT301D2 and BdUGT429A1,in which BdCncC,BdMaf-K alone and BdCncC/BdMaf-K combination could induce an increase in fluorescence activity in promoter region of BdUGT301D2.While for BdUGT429A1,only the combined use of BdCncC/BdMaf-K could regulate its upregulated expression.The key transcription factor binding sites of BdUGT301D2 and BdUGT429A1 were located between-441 ～ 0 and-367 ～ 0,respectively.Subsequently,using RNAi and Western Blot,it was found that after silencing BdMaf-K alone(silencing efficiency was 61.10%),the expression of BdUGT301D2 decreased by 70.62%,while the expression of BdUGT429A1 didn’t change,and the susceptibility to malathion increased by 16.94%.While silencing BdCncC alone(silencing efficiency was 79.93%)reduced the expression of BdUGT301D2 and BdUGT429A1 by 90.05% and 52.55%,respectively,and increased the susceptibility to malathion by 18.20%.Finally,the combined silencing of BdCncC/BdMaf-K(silencing efficiency was 49.23% and 64.51%,respectively)reduced the expression of BdUGT301D2 and BdUGT429A1 by 90.80% and 64.51%,respectively,and increased the susceptibility to malathion by 21.80%.The results indicated the different mechanism of transcriptional regulation between BdUGT301D2 and BdUGT429A1,in which BdUGT301D2 is regulated by both BdMaf-K homodimer and BdCncC/BdMaf-K heterodimer,whereas BdUGT429A1 was only regulated by BdCncC/BdMaf-K heterodimer.Taken together,the BdCncC/BdMaf-K pathway regulates the overexpression of BdUGT301D2 and BdUGT429A1 in MR and mediates the resistance to malathion of B.dorsalis.5 Reactive oxygen species(ROS)activate BdCncC/BdMaf-K pathway to regulate downstream target UGT genesUsing the ROS inhibitor NAC to feed MR strains,the expression of BdCncC,BdUGT301D2 and BdUGT429A1 decreased by 38.91%,76.80% and 35.18%,respectively,accompanied by increased susceptibility to malathion.However,NAC treatment had no significancely affect in MS strains.Using the ROS activator curcumin to feed MR strain,the expression levels of BdCncC,BdUGT301D2 and BdUGT429A1 in MR and MS strains were increased by 0.79-,0.99-,0.60-fold and 5.86-,0.94-and1.04-fold,respectively.At the same time,the susceptibility of the two strains to malathion decreased significantly.These results suggested that ROS might affect the nuclear translocation of BdCncC,and then affect the regulation of BdCncC to BdUGT301D2 and BdUGT429A1 in mediating malathion resistance of B.dorsalis.Based on RT-qPCR,the expression of SOD2,Duox,Nuox,and CAT were overexpressed1.38-to 31.60-fold in the MR strain.It was found that the enzyme activities of SOD,CAT,T-AOC,MDA and GST up-regulated with 1.09-to 1.90-fold in MR strain compared to that in MS strain,but there was no difference in the enzyme activity of POD.All the above results indicated that ROS content was significantly higher in the MR strain than in the MS strain of B.dorsalis.In conclusion,the increased ROS content of B.dorsalis activates BdCncC/BdMafK pathway to regulate the up-regulation of BdUGT301D2 and BdUGT429A1,which mediates the enhanced malathion resistance of B.dorsalis.The results of this thesis clearly define the functions and regulatory mechanisms of UGTs as secondary detoxifying metabolic enzymes in the formation of insect metabolic resistance in theory,while also providing a theoretical basis for controlling and preventing B.dorsalis in the field. |
PDF Full Text Request