Molecular Characteristics And Functions Of CYP6CV1 And CYP6AB51 Genes In Conogethes Punctiferalis

The polyphagous Conogethes punctiferalis(Lepidoptera: Pyralidae)is an important economic pest that seriously damages fruit trees and agricultural crops.In recent years,the damage of C.punctiferalis to summer maize has gradually increased,and it has become the dominant species of summer maize pests in the Huanghuaihai area.Studies have shown that there are a large number of detoxification enzymes in insects,they play an important role in the process of insects resisting foreign substances and maintaining their normal growth and development.Cytochrome P450(CYP450)enzymes are an important class of detoxification enzymes in insects.When insects face the stress of toxic substances,their activity and transcription levels will increase,thereby degrading and metabolizing the toxic substances into less toxic forms,thereby improving the adaptability of insects to unfavorable environments.The overexpression of this cytochrome P450 enzyme gene is also one of the main reasons for insect resistance.We obtained two cytochrome P450 genes,CYP6CV1 and CYP6AB51,from the previous transcriptome sequencing results of the research group.In this study,the molecular characteristics of the two target genes were studied by gene cloning and amino acid bioinformatics analysis.The transcriptional patterns of CYP6CV1 and CYP6AB51 genes and their functional roles in adaptation to three commonly used chemical pesticides were analyzed by quantitative real-time PCR(q RT-PCR)and RNA interference(RNAi)techniques.The relevant research results are as follows:(1)The c DNA sequences of CYP6CV1 and CYP6AB51 were obtained by gene cloning and sequencing,and their open reading frames were 1503 bp and 1536 bp,respectively.At the same time,amino acid bioinformatics analysis showed that CYP6CV1 and CYP6AB51 of C.punctiferalis encode 500 and 511 amino acids,respectively,with protein molecular weights of 57.045 and 59.343 k Da,and isoelectric points of 8.93 and 8.38,respectively.Their sequences were submitted to NCBI and obtained Gen Bank accession numbers MT740277.1 and MW402840.1,respectively.Phylogenetic analysis showed that CYP6CV1 and CYP6AB51 of C.punctiferalis were closely related to Cnaphalocrocis medinalis CYP6CV1 and Chilo suppressalis CYP6AB51,respectively,with amino acid homology of 78.45% and 61.36%,respectively.The predicted amino acid sequences encoded by the two genes contained five typical conserved structural regions of insect P450 proteins,a C-helix region(WXXXR),I-helix region(AGXETS),K-helix region(EXXR),meander sequence(PXXFXPXXF)and heme-binding sequence(FXXGXRXCXG)(where X represents any amino acid).(2)qRT-PCR was used to detect the gene expression levels of CYP6CV1 and CYP6AB51 at different larval developmental stages and in different tissues of C.punctiferalis.The two target genes were expressed throughout the C.punctiferalis larval growth period and in different tissues.As the larvae developed,the expression levels of the two genes increased.The maximum expression levels of CYP6CV1 and CYP6AB51 genes were detected in the fifth-instar larvae stage,and they were 385.96 and 12.42 times higher than in the first-instar larvae,respectively.Among the different tissues,the expression levels of the two genes in the midgut were the highest,which were significantly higher than in other larval tissues,and the transcript levels were 11.60 and156.96 times higher than in the head,respectively.(3)The toxicity of the three chemical insecticides,chlorantraniliprole,emamectin benzoate and lambda-cyhalothrin,on C.punctiferalis larvae by bioassays.The C.punctiferalis larvae were susceptible to the three tested pesticides,and their LC50 were0.2028 μg/ g,0.0683 μg/g and 0.6110 mg/L,respectively.(4)Three pesticides commonly used in the control of C.punctiferalis were selected to evaluate their effects on the expression levels of C.punctiferalis CYP6CV1 and CYP6AB51 as assessed by q RT-PCR.The LC10 value of chlorantraniliprole was treated for 3 h,the induction effect on CYP6CV1 was the strongest,and the LC30 value of 6 h treatment was the strongest induction effect on CYP6AB51.The LC30 value of emamectin benzoate treatment for 24 h showed the strongest induction effect on the two target genes.The LC10 value of lambda-cyhalothrin for 24 h had the strongest induction effect on CYP6CV1,and the LC30 value treatment for 3 h had the strongest induction effect on CYP6AB51.(5)The functional roles of CYP6CV1 and CYP6AB51 in C.punctiferalis were further studied by RNAi.After the silencing of CYP6CV1 and CYP6AB51 independently in C.punctiferalis by injecting ds CYP6CV1 and ds CYP6AB51,respectively,the expression levels of CYP6CV1 and CYP6AB51 showed maximum silencing at 36 h relative to the control groups,with reductions of 72.91% and 70.94%,respectively.After ds RNA injection,the mortality of CYP6CV1-silenced larvae and CYP6AB51-silenced larvae treated independently with LC10 values of chlorantraniliprole,emamectin benzoate and lambda-cyhalothrin were recorded.The results indicated that the delivery of ds CYP6CV1 increased the larval mortality caused by chlorantraniliprole(from 1.67% to 11.67%),emamectin benzoate(from 3.33% to 16.67%)and lambda-cyhalothrin(from 3.33% to13.33%),and injection with ds CYP6AB51 significantly increased the larval mortality caused by chlorantraniliprole(from 1.67% to 8.33%),emamectin benzoate(from 3.33%to 11.67%)and lambda-cyhalothrin(from 3.33% to 11.67%).