Functional Analysis Of CYP4G199 Gene In Integument Development Of Mythimna Separata

Mythimna separata is included in the list of crop diseases and insect pests,causing serious economic losses when it occurs,and it is unremitting in the prevention and control.With the use of pesticides,its resistance is becoming increasingly serious,so the resistance control and green prevention and control of M.separata have become a research hotspot.Cytochrome P450(CYP)is closely related to the growth and development of insects and the metabolism of endogenous and exogenous substances.More and more studies have found that P450 family genes are involved in the synthesis of cuticular hydrocarbons,resulting in enhanced resistance to insecticides.In this study,based on the important function of P450 gene in integument development,the transcriptome data of M.separata integument were obtained by transcriptome sequencing,and the specific high expression of CYP4G199 gene in integument was obtained by screening and identification.The spatio-temporal expression pattern of this gene in M.separata was explored.The RNAi technology system of M.separata was successfully established to verify the function of CYP4G199 in M.separata integument development.The results provide a theoretical basis for the development of P450 inhibitors or insecticides and the search for green prevention and control methods of M.separata.The main research results are as follows:1.Sequencing of integument transcriptome and identification of P450 gene in M.separataThrough transcriptome sequencing,166 transcripts of P450 gene and 4 transcripts of cytochrome P450 reductase(CPR)gene were identified from the integument of 4th instar larvae.Among them,66 P450 genes and 1 CPR gene had complete open reading frame(ORF).These genes included four groups(CYP2,CYP3,CYP4 and Mitochondrial).They are mainly distributed in the CYP4,CYP6,CYP9 and CYP340 families,all of which contain Helix C,Helix I,Helix K and heme-binding domains.2.Screening the P450 gene with high specific expression in M.separata integumentBased on FPKM(Fragments Per Kilobase Per Million mapped Fragments)of transcriptome data,ten P450 genes were identified,including CPR gene.Further verification of the relative expression level of these 10 genes in the integument was consistent with the transcriptome sequencing data.The expression level of CYP4G199 gene was significantly higher than that of other genes.RT-qPCR was used to detect the expression levels of these 10 genes in different tissues of larvae.It was found that five genes,such as CYP4G199,were specifically expressed in larvae.Therefore,CYP4G199 gene is highly expressed in integument.3.Cloning and sequence analysis of CYP4G199 geneThe cDNA sequence of CYP4G199 gene was cloned,which contained 1695 bp ORF and encoded 564 amino acids,including the P450 protein characteristic sequence of Helix C,Helix I,Helix K and heme-binding domain.Based on genomicDNA,the length of DNA sequence of CYP4G199 was 6985 bp,including 9 introns,the length of which was 331-1538 bp,and the length of exon was 70-238 bp.The junction of each intron and exon conformed to GT-AG principle.It was found that CYP4G199 and CYP4G74 had the highest homology,and the coincidence rate reached 92.32%.4.Analysis of the spatiotemporal expression pattern of CYP4G199 gene by RT-qPCRRT-qPCR was used to detect the expression of CYP4G199 in different developmental stages of M.separata.It was found that the expression level of CYP4G199 was the highest in the first instar larva.With the growth of larval ininstar,the expression level of CYP4G199 gene gradually decreased,and the expression level of females was slightly higher than that of males in adult stage.By comparing the expression levels of CYP4G199 in the integument of larvae of different instars,it was found that CYP4G199 was expressed in the integument of all instar larvae.The expression level of CYP4G199 in the integument of larvae of the first instar was relatively high,the expression level of CYP4G199 was relatively high in the integument of 1st instar larvae,but low in the integument of 6th instar larvae.There was no significant difference in the expression levels of CYP4G199 in the integument of 2nd instar to 5th instar larvae.The expression level of CYP4G199 gene in the integument was the lowest at 6 h after molting stage,and increased gradually with the end of molting.At the beginning of the 4th instar,the expression level of CYP4G199 gene in the integument was not significantly different from that in the early molting stage,which indicated that the expression level of CYP4G199 increased significantly during the process of old integument digestion and new integument formation.5.Study on the function of CYP4G199 gene in the development of M.separata integument by RNAiEstablish the RNAi technology system of M.separata,through the in vitro synthesis of ds RNA,microscopic injections to RNAi 4th instar larvae,the results showed that the expression of CYP4G199 gene was inhibited,and its expression was significantly down-regulated by 65%.In addition,the integument development of the 4th instar larvae was abnormal from molting to the 5th instar larvae,and the 6th instar larvae were unable to normalize pupae.This suggests that CYP4G199 is related to insect integument development and may be involved in integument synthesis.