The Mechanism Of Transcriptional Regulation Of The Cytochrome P450 CYP6ER1 Responsible For Nitenpyram Resistance In Nilaparvata Lugens (St(?)l)

Nilaparvata lugens is an important pest in rice production in China,and chemical control is the main methods to suppress N.lugens.Nitenpyram is an insecticide as a nicotinic acetylcholine receptor(nAChR)competitive modulator in insect pests,and is a neonicodinods insecticide mainly acting against sucking pests,such as Nilaparvata lugens(St(?)l)and Sogatella furcifera.Currently,the field populations of N.lugens is medium to high levels resistance to neonicotinoids such as nitenpyram.Previous studies showed that the resistance of N.lugens to nitenpyram was caused by the overexpression of P450 gene CYP6ER1,but the transcription regulation mechanism of CYP6ER1 gene was not well understood.In this study,the role of CYP6ER1 gene in N.lugens resistance to nitenpyram was explored,and the promoter sequence of CYP6ER1 gene was analyzed from the perspective of transcription regulation,so as to select transcription factors that could regulate CYP6ER1 gene and study the relationship between transcription factors and nitenpyram resistance.The main results are as follows:1.Based on the NCBI database,the N.lugens strain NR was amplified by PCR,and the upstream sequence of P450 gene CYP6ER1 was obtained.Seven expression vectors with different lengths were constructed.Through the double luciferase reporting experiment,it was found that there was a promoter core region in the upstream of the promoter from 1160 to 1352 bp.After sequence analysis,nine transcription factors were screened out.Transcription factors were cloned into PIEXHis vector and co-transfected with the promoter fragment.It was found that Cncc and Paxillin had certain promotive effects on promoter.2.The expression of transcription factors in laboratory resistant strain NR and susceptibile strain NS was detected by qRT-PCR.The results showed that Cncc,AP-1,Paxillin and Pou were up-regulated in resistant population.These four genes may be involved in the resistance of N.lugens to nitenpyram.The expression level of transcription factors of Adf-1,AKH,HNF4,Maf and CEBP was down-regulated in resistant population.By treating NR and NS strains with different concentrations of nitenpyram,the variation of transcription factor expression was detected.The results showed that the expression levels of transcription factors Adf-1,AKH,CEBP,HNF4,Maf,AP-1,Paxillin and Pou in NR strains were inhibited to a certain extent with the ingestion of insecticides by N.lugens.Then,with the increase of time,the expression level would be gradually upregulated,and then decreased again.Except Adf-1,AKH and Cncc,the expression changes of other transcription factors in NS strain were inhibited3.The dsRNA primers were designed for four transcription factors,and the synthesis of dsRNA was induced by Escherichia coli HT115.The stability of dsRNA in sucrose solution was tested.The results showed that dsRNA was stable and not degraded when placed in 27 ℃ incubator for 48 h.The RNAi of transcription factors was performed by an artificial feeding method,and the expression changes of transcription factors and CYP6ER1 were detected by qRT-PCR.The results showed that the silencing of Cncc and Maf was positively correlated with the expression of CYP6ER1 gene,and the interference of these two transcription factors may inhibit the expression of CYP6ER1 gene.The bioassay results showed that the mortality of N.lugens resistant strains fed on ds AKH,ds HNF4,ds Cncc,ds Maf had no significant change with that of N.lugens resistant strains fed on ds GFP.The mortality rate of N.lugens susceptible strain fed on ds HNF4,ds Cncc,ds Maf was significantly different from that of N.lugens susceptible strain fed on ds GFP.These results indicated that HNF4,Cncc and Maf could be involved in the resistance of N.lugens to nitenpyram to a certain extent.