Resistant Mechanism Of Cytochrome P450 Mediated Sitobion Miscanthi On Imidacloprid

Sitobion miscanthi is one of the dominant species that harm wheat.For a long time,its control has been mainly based on chemical pesticides.The main pesticides include organochlorines,organophosphates,carbamates,and pyrethroids.However,due to the continuous and large-scale use of chemical pesticides,wheat aphids have exerted a great selection pressure on insecticide resistance,which in turn led to different levels of resistance to commonly used pesticides in wheat aphids in various regions.In this paper,Sitobion miscanthi was the research object,the differentially expressed cytochrome P450 genes were screened by transcriptome sequencing technology.Using the experimental technology of real-time fluorescent quantitative PCR（qPCR）and RNA interference（RNAi）to verify the main P450 genes.Then cloning the main P450 genes 5’untranslated region（5’UTR）sequence,detecting promoter activity,and slenceing transcription factor experiments were analyzed to reveal the P450-mediated Sitobion miscanthi resistance mechanism to imidacloprid.The results are as follows:1.The contact toxicity of imidacloprid on the Sitobion miscanthi third instar was measured by leaf-dipping with aphids method.The results showed that the LC₅₀ of imidacloprid was 1.341 mg/L and LC₁₀ was 0.506mg/L.2.After treatment with imidacloprid(LC₁₀)for 24 h,the differentially expressed cytochrome P450genes were screened out by transcriptome sequencing technology.The results showed that there were 108differentially expressed P450 genes,of which 37 P450 genes were up-regulated and 71 P450 genes were down-regulated.3.Analyzing the differential expression of two P450 genes CYP6DD1 and CYP307A2 in imidacloprid resistant stains（SM-R）and sensitive stains（SM-S）by qPCR.The results showed that the expression levels of the two P450 genes in the SM-R strain were significantly higher than those in the SM-S strain,which were 2.23 and 3.16 times that of the SM-S strain,respectively.4.After feeding on ds CYP6DD1 and ds CYP307A2 for 24 h,qPCR was used to detect the silencing efficiency.The results showed that compared with the control（dsGFP）,the silencing efficiency of CYP6DD1 and CYP307A2 genes reached 36.3%and 45.3%,respectively.After feeding ds CYP6DD1 and ds CYP307A2 for 24 h,and then treating with imidacloprid(LC₁₀)for 24 h,the death rate of Sitobion miscanthi was 1.87 and 2.09 times that of the control group（dsGFP）,respectively.5.In order to explore the mechanism of P450 genes expression regulation,a full-length two P450genes were cloned and their promoters were identified from Sitobion miscanthi.Transient transfection results showed that the recombinant vectors of p HN5,p HN4 and p HN3 of CYP6DD1 and p HN5,p HN4 and p HN3of CYP307A2 were effective,their promoter activity was significantly higher than the control p GL4.11.Through bioinformatics softwares predict that there are binding sites of the transcription factor Nrf2 at-344to-338 bp and+159 bp to+165 bp of CYP6DD1,and+103 bp to+113 bp of CYP307A2,respectively.After silencing the Nrf2 gene by RNAi,the expression of CYP6DD1 and CYP307A2 decreased by 56.4%and45.1%,respectively.After feeding ds Nrf2 for 24 h,then treating with imidacloprid(LC₁₀)for 24 h,the death rate of Sitobion miscanthi was 2.87 times that of the control group（dsGFP）.