Identification Of UDP- Glycosyltransferases Conferring Resistance Of Thiamethoxam In Aphis Gossypii

UDP-glycosyltransferases(UGTs)are major phase II enzymes of a detoxification system evolved in all kingdoms of life.UGTs conjugate a variety of small lipophilic molecules with sugars and alter them into more water-soluble metabolites.Therefore,glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds.Thiamethoxam is a second-generation nicotinic high-efficiency and low-toxic insecticide with chlorothiazole structure,which irreversibly binds to the nicotinic acetylcholine receptor(nAChR)of insect nervous system cells and interferes with the transmission of insect nerve impulses.Eventually,the insects are paralyzed to death.Aphis gossypii,a worldwide pest with broad-spectrum resistance to insecticides,The role of UGTs in the detoxification and metabolism of thiamethoxam is not clear?In this study,two inhibitors of UGT enzymes,sulfinpyrazone and 5-nitrouracil,significantly increased the toxicity of thiamethoxam against the resistant strain of Aphis gossypii,which indicates that UGTs are involved in thiamethoxam resistance in the cotton aphid.Based on transcriptome data,31 A.gossypii UGTs belonging to 11 families(UGT329,UGT330,UGT341,UGT342,UGT343,UGT344,UGT345,UGT348,UGT349,UGT350,and UGT351)were identified.Compared with the thiamethoxam-susceptible strain,the transcripts of 23 UGTs were elevated,and the transcripts of 13 UGTs(UGT344J2,UGT348A2,UGT344D4,UGT341A4,UGT343B2,UGT342B2,UGT350C3,UGT344N2,UGT344A14,UGT344B4,UGT351A4,UGT344A11 and UGT349A2)were increased by approximately 2.0-fold in the resistant cotton aphid.Five UGT genes were successfully knocked out by feeding cotton aphid dsRNA,UGT348A2,UGT344B4 and UGT344J2 were able to significantly increase the sensitivity of cotton aphid to thiamethoxam after being silenced.Based on real-time fluorescence quantitative results and transcriptome expression level,we cloned UGT344B4,UGT344J2 gene for prokaryotic expression in vitro.Among them,UGT344J2 was successfully expressed and active.The enzymatic reaction of UGT344J2 protein about the specific substrate thiamethoxam was detected by Liquid chromatography system.It was found that UGT344J2 protein could not promote the glycosylation of thiamethoxam.