| Bemisia tabaci,as a world-wide euryphagous pests,annually cause huge loss in agriculture and economy all over the world due to its comprehensive feeding habits,fast multiplication,capacity to spread plant virus and cause sooty blotch.Methods to control B.tabaci include physical control,biological control and chemical control,and chemical control is still the main method.Among that,neonicotinoids are widely applied in control B.tabaci,due to its specific mechanism,harmless character to environment and mammal.However,pesticide abuse has made B.tabaci develop a series of resistance mechanism under the pesticide selection pressure,its resistance is widely reported all over the world and developing annually,which take challenge to human’s agricultural pursuits.Insects build resistance mainly by detoxification enzymes in vivo.Among that,cytochrome P450 enzymes,as phase I detoxification enzyme,are reported to mediate resistance and studied in several species.Cytochrome P450 enzyme is a vast family which plays a detox role by oxidating,hydrolyzing and reducing pesticide inside insects.To date,a large number of studies have indicated the connection between increased expression of P450 genes and resistance to many insecticides in pests;these connections are usually based on overexpression or allelic mutation of one or multiple P450 genes.Insecticide resistance of B.tabaci has been widely reported in previous study,however,the underlying mechanism reminded unclear.In this study,we choose P450 genes,CYP6DW3 and CYP6DW5v1,which were extremely overexpressed in imidacloprid resistant strain based in transcriptome as the research subject,and study them in indoor and field B.tabaci populations,finally wo reveal that CYP6DW3 and CYP6DW5v1 mediate B.tabaci imidacloprid resistance,the main research results as follows:1.After positive and reverse screening and rearing indoor strain,we have acquired indoor susceptible and resistant strains which shared same genetic background,meanwhile we have sampled in field to acquire field susceptible and resistant strains as the research basics.In addition,we have monitored their imidacloprid resistance level.After being exposed in imidacloprid for ten years,the resistance level of indoor resistant strain has reached to about 20,for field populations,their resistance level are among 1.14 to 29.27.2.According to transcriptomes,we have figured out the basic information ofCYP6DW3 and CYP6DW5v1,they both belong to the P450 gene superfamily and are found the common features of P450 gene.By build phylogenetic tree,we have found that the P450 s of B.tabaci clustered with those of Hemiptera.Besides,after gene expression quantification of B.tabaci different developmental stages and tissues by q RT-PCR,we build spatiotemporal expression profile of target gene.3.We successfully clone two P450 genes in indoor susceptible and resistant strains,the sequence alignment results showed that no stable base mutation exist in them.These genes were markedly overexpressed in imidacloprid-resistant B.tabaci populations compared with susceptible populations,among that up to 9 times and 11 times respectively.To verify function of these two P450 genes,we knockdown their expression level by feed indoor resistant strain specific double-strain RNA,the results showed that knockdown of these genes decreased the imidacloprid resistance of B.tabaci,similar results were acquired in field resistant strains.Moreover,heterologous expression of B.tabaci P450 genes in SF9 and H5 cells and metabolic studies showed that the CYP6DW3 protein could metabolize imidacloprid and produced imidacloprid-urea in vitro.Collectively,CYP6DW3 and CYP6DW5v1 are markedly overexpressed in indoor and field imidacloprid resistant B.tabaci strain,and the expression levels of CYP6DW3 and CYP6DW5v1 are positively correlated with resistance level in Bemisia tabaci.Our study further reveals that cytochrome P450 enzymes affect the physiological activities related to resistance in insects,which helps scholars more deeply understand resistance mechanism and contributes to the development of integrated pest management framework in theory. |
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