Identification Of Cytochrome P450 Genes Related To Chlorantraniliprole Resistance In Mythimna Separata(Walker)

Mythimna separata（Walker）is a migratory,polyphagous,explosive,worldwide pest that has developed resistance to a variety of pesticides.This study selected M.separata as the object,the third instar larvae treated with chlorantraniliprole or fipronil for 24 hours,construction of transcriptome sequencing library and RNA seq were conducted,Then the internal reference genes selected,differentially expressed P450 genes validated by real-time fluorescent quantitative PCR,and RNA interference（RNAi）was explored to confirm the main P450 genes related to detoxification of insecticides.The detailed results of the study are as follows:1.The leaf dipping method was used to determine the virulence of chlorantraniliprole and fipronil for controlling the M.separata.The results showed that the LC₅₀0 of chlorantraniliprole or fipronil to the third instar larvae of M.separata was 0.45 and 18.72mg/L respectively,and the LC₁₀0 to the third instar larvae of M.separata was 0.15 and 13.66mg/L,respectively.2.The third instar larvae of M.separata were treated with chlorantraniliprole(LC₁₀)or fipronil(LC₁₀)for 24 hours,construction of transcriptome sequencing libraries and RNA seq.29 P450 genes were up-regulated and 27 P450 genes were down-regulated in the chlorantraniliprole treatment;23 P450 genes were up-regulated and 26 P450 genes were up-regulated in the fipronil treatment.3.The 8 commonly used as internal reference genes,includingβ-actin（β-ACT）,18s ribosomal（18S）,28s ribosomal（28S）,glyceraldehyde-3-phosphate（GAPDH）,elongation fator-alpha（EF1-α）,TATA box binding protein（TBP）,ribosomal protein L7（RPL7）,alpha-tubulin（α-TUB）were selected to confirm the suitable reference under different conditions.We found that the most suitable reference genes for the different experimental conditions by qPCR and the programs of△Ct,geonorm,NormFinder and BestKeeper.For developmental stages,28S/RPL7 were the optimal reference genes;For different tissues,RPL7/EF1-αwere the optimal reference genes;whereas for insecticide treatments,28S/α-TUB were the optimal reference genes.4.The 12 P450 genes affected by chlorantraniliprole or fipronil were screened from transcriptomic library,and the differential expression of P450 gene were verified by fluorescence quantitative PCR.The results showed that the expression levels of 7 P450 genes（CYP321A10,CYP4M6v2,CYP18A1-1,CYP301A1-2,CYP6B1-1,CYP333A1-1 and CYP6B1-2）were significantly up-regulated after chlorantraniliprole treatment,which were2.22,2.54,2.87,3.62,3.86,4.35 and 5.63 times that of the control.The expression levels of the 5 P450 genes（CYP6B42,CYP6B6v2,CYP6AB32,CYP6AN1 and CYP9A11）were significantly down-regulated compared with the control,which were 3%,9%,18%,26%and28%,respectively.After the fipronil treatment,the expression levels of the 4 P450 genes（CYP6B1-2,CYP18A1-1,CYP6B1-1 and CYP301A1-2）were significantly up-regulated compared with the control,which were 1.75,2.38,2.63 and 4.57 times of the control,respectively.The expression levels of the 5 P450 genes（CYP9A11,CYP6AB32,CYP6B6v2,CYP6AN1 and CYP6B42）were significantly decreased compared with the control,which were 10%,13%,19%,21%and 63%,respectively.The expression levels of CYP321A10,CYP4M6v2 and CYP333A1-1 were not significantly different from those of the control group.5.4 P450 genes,including CYP18A1-1,CYP6B1-1,CYP6B1-2 and CYP301A1-2 were silenced by RNAi technology.The results showed that the silencing efficiency of CYP18A1-1,CYP6B1-1,CYP6B1-2 and CYP301A1-2 was 46%,54%,63%and 53%after 48 h,respectively;After feeding dsRNA for 24 h,LC₁₀0 chlorantraniliprole was added to the feed,and then fed for 24 h,the sensitivity of silenced P450 genes to chlorantraniliprole was detected,and the mortality of the 4 P450 genes（ds CYP18A1-1,dsCYP6B1-1,dsCYP6B1-2and dsCYP301A1-2）after treatment were 1.71,1.86,2.43 and 2-flod compared to the control group（dsGFP）.