Identification Of Abc Transporter Genes Account For Spirotetramat Resistance And Their Transcriptional Regulation In Aphis Gossypii Glover

Cotton aphid（Aphis gossypii Glover）was the global agricultural pest with the most complex insecticide resistance background.Spirotetramat was the tetramic acid derivate targeting the acetyl-Co A carboxylase（ACC）,blocking the normal energy storage of insects causing nymph death and reducing adult fecundity.Although there were few cases of spirotetramat resistance in the field strains of cotton aphids,continuous application may accelerate the development of spirotetramat resistance.Therefore,in-depth research on the potential resistance mechanisms of novel insecticides can provide theoretical basis for delaying the development of insecticide resistance in field populations.The main results of this study are as follows:ABC transporter inhibitors（verapamil,MK-571,NSC23925 and tectochrysin）were used for synergistic bioassay and fluorescence elimination experiments on ABC transporter substrates（Texas Red）,exploring the role of ABC transporters in the resistance of cotton aphids to spirotetramat.Synergism bioassays showed 17.05%and35.42%increases in the toxicity to spirotetramat with the ABC inhibitor verapamil in adult and 3rd instar nymph aphids of the SR strain,respectively.In the fluorescence elimination experiments,verapamil（general ABC inhibitor）and spirotetramat significantly inhibited the elimination of Texas Red.However,the other three ABC transporter inhibitors failed to inhibit the elimination of Texas Red.RNA was extracted from the body（RC）and midgut（MG）tissues of cotton aphids for transcriptome analysis.A total of 71 ABC transporter genes were identified from the transcriptome.We screened 11 ABC transporter genes which were upregulated or had high expression abundance for further research.The results of RT-q PCR showed that ABCF2,MRP11 and MRP12 were specifically upregulated in the midgut of the SR strain.Compared with the SS strain midgut,the transcription levels of ABCB4,ABCB5,ABCD1,ABCF2,ABCG17,MRP11,and MRP12 in the SR strain midgut were significantly upregulated.Using UAS/GAL4 transgenic Drosophila to ectopically expressed ABC transporter genes,the bioassay results showed that the ABCB4-,ABCB5-,ABCF2-,MRP11-and MRP12-overexpressing flies significantly increased the tolerance of spirotetramat.Moreover,the upregulated ABCF2,ABCB4,and ABCB5 in the midgut contributed more to the resistance of spirotetramat in in vitro functional analysis.The bioassay results showed that the mortalities of SR strain under treatment with spirotetramat were significantly increased by 1.71,1.65,1.58,1.81 and 1.83 folds after the suppression of ABCB4,ABCB5,ABCF2,MRP11 and MRP12,respectively.Molecular docking of spirotetramat to ABC transporters indicated hydrogen bond interactions,which indicate that the ligand docked to the ABCB4,ABCB5,ABCF2,MRP11 and MRP12 proteins with similar binding energies（<-6.90～-8.30 kcal/mol）.According to the predictions of TRANSFAC7.0 and JASPAR database,the binding sites of CROC,BCD,CF2-II or BRC-Z4 were located in the promoters of ABCB4,ABCB5,ABCF2,MRP11 and MRP12.Yeast one-hybrid（Y1H）results showed that transcription factor CF2-II（C₂H₂ zinc finger protein）can bind with the promoters of ABCF2,ABCB5 and MRP12.The expression levels of ABCF2,ABCB5 and MRP12were significantly upregulated after silencing CF2-II.The results of dual-luciferase reporter assays demonstrated a negative regulatory relationship between CF2-II and ABC transporter promoters.Furthermore,Y1H and electrophoresis mobility shift assays（EMSAs）revealed that CF2-II inhibited the expression of ABC transporter genes through interaction with binding sites[ABCF2.p（-1149/-1140）or MRP12.p（-1189/-1181）].In summary,this thesis used transcriptome and RT-q PCR to screen differentially expressed genes related to spirotetramat resistance,and combined multiple molecular biology experimental techniques to explore the relationship between spirotetramat resistance and the upregulated ABC transporter genes in the midgut.Clarify the expression and regulation mechanism of resistance related ABC transporter genes.The research results of this thesis have successfully revealed the role of ABC transporters in the formation of spirotetramat resistance in cotton aphid,providing a theoretical basis for future cotton aphid control.