| Aphis gossypii Glover is one of the most economically important insect pests in agriculture.With the characteristics of strong reproductive capacity,short growth cycle and alternation of generations,it has developed resistance to common insecticides.In this study,cotton aphid with resistance to thiamethoxam,which has been screened in laboratory for many years,was used as experimental material.Synergism analysis has indicates that P450 s are involved in thiamethoxam resistance.In this study,expression changes in the transcripts of P450 genes were determined in thiamethoxam-susceptible and thiamethoxam-resistant strains.It provides a theoretical basis for delaying the development of resistance level of cotton aphid and prolonging the service life of thiamethoxam.Cytochrome P450 monooxygenases represent a key detoxification mechanism in neonicotinoids resistance in cotton aphid.The expression levels of P450 genes were tested by RT-q PCR in the susceptible and resistant strains of cotton aphid to thiamethoxam.Nine P450 genes(CYP6CY14,CYP6DC1,CYP6CZ1,CYP6DD1,CYP6CY9,CYP6CY13-2,CYP6CY5,CYP6CY18 and CYP6CY12)in CYP3 clade were significantly overexpressed in the resistant strain compared with the susceptible strain,especially CYP6CY14,which was increased 17.67-fold.Transcripts of ecdysone synthesis-related P450 genes,including CYP302A1,CYP306A1,CYP307A1 and CYP315A1,were up-regulated in the resistant strain,which may accelerate molting hormone production.The ecdysone response genes(ecdysone receptor(Ec R),ultra-spiracle(USP)and Broad-complex protein(Br-C)were overexpressed in the resistant strain.RNA interference(RNAi)targeting CYP6CY14 significantly increased the sensitivity of the resistant aphid to thiamethoxam.Artificial nutrient solution containing ds RNA were fed to the aphids.The m RNAs targeting CYP6CY14 interfered(RNAi)to 0.42 fold.The mortality of resistant cotton aphids ranged from 26.43% of the control to 59.86% of the aphids fed by ds RNA-CYP6CY14.In the early stage of the experiment,the 5' flanking sequence upstream of CYP6CY14 was cloned by Genome walk,and the transcription factors were analyzed and predicted.By targeting knockdown the related transcription factors,the results showed that the expression levels of CYP6CY14 decreased by 0.22,0.50,0.75,respectively,and the expression levels of Dfd,Abd-A,and zen decreased to 0.57,0.20,and 0.22,respectively.The experimental results indicated that transcription factors of Dfd,Abd-A and zen may be involved in the transcriptional regulation of CYP6CY14 promoter in cotton aphid.However,the 5 'side chain activity of CYP6CY14 was detected to be very low by cell transfection experiments,and the core sequence of the promoter may not located in the upstream sequence of the 5' side chain of promoter CYP6CY14.Based on the genome data of cotton aphid,suitable fragments of 5' upstream of CYP6CY14 were searched in Gen Bank.Three transcription factors,Abd-A,Dfd and zen,were found to have binding sites at the upstream flanking sequence 5 'of CYP6CY14.It has greatly supported the former results of the transcriptional regulation to CYP6CY14,which provides a theoretical basis for the verification of subsequent transcription factors and promoter activity analysis.The results of the present study indicate the possible involvement of these P450 genes in thiamethoxam resistance and the transcriptional regulation to CYP6CY14.These results are useful for understanding the mechanism of thiamethoxam resistance and will contribute to the management of insecticide-resistant cotton aphids in China. |
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