Cloning And Functional Studies Of CYP4G15 And CYP4l4 Genes In Spodoptera Frugiperda

Cytochrome P450 is the oldest and largest supergene family.As one of the most important detoxification enzymes in insects,it is not only closely related to the synthesis of20-hydroxyecdysone,juvenile hormone and pheromone,but also plays a very important role in insect metabolism of pesticides and plant secondary metabolites.Spodoptera frugiperda is a polyphagous insect distributes in the tropical and subtropical areas of North and South America,belongs to the genus frugiperda（Lepidoptera:Noctuidae）.It has been one of the most difficult agricultural pests to be controlled because of its strong migration ability,reproduction ability,adaptability and resistance.In the process of using chemical methods to control S.frugiperda,the problem of resistance to insecticides must be overcome.The degenerate primers were designed by comparing the gene sequences of CYP4G15 and CYP4L4 of known insects.The full length sequences of the two genes were successfully cloned by PCR amplification method.The expression differences and distribution of CYP4G15 and CYP4L4 in different developmental stages and different tissues were detected by RT-q PCR method,as well as the expression levels of CYP4G15 and CYP4L4 under the stress of chlorfenapyr and indoxacarb.In addition,the molecular mechanism of CYP4G15 and CYP4L4 genes in response to insecticide stress was explained by RNAi method,In order to solve the important scientific problem of insecticide resistance of S.frugiperda.and the following research results were obtained:1.Cloning and spatiotemporal expression of CYP4G15 and CYP4L4 in S.frugiperda.The full-length sequences of CYP4G15 and CYP4L4 were cloned by PCR method.The results showed that the CYP4G15 open reading frame was 1692 bp,encoding 563 amino acids,and the CYP4L4open reading frame was 1479 bp,encoding 492 amino acids.RT-q PCR was used to detect the expression of the two genes in different developmental stages and different tissues.The results showed that the expression levels of both genes were highest in the young larvae of S.frugiperda.and the two genes were speculated to play an important role in the development of the young larvae of S.frugiperda.The expression level of CYP4G15 was the highest in the epidermis tissue,and the expression level of CYP4L4 was the highest in the foregut tissue,which may indicate that they perform different functions in different tissues.2.Toxicity of insecticide mixtures against S.frugiperda and expression of CYP4G15 and CYP4L4 under insecticide stress.Toxicity tests were carried out in laboratory on the 3^rdinstar larva of S.frugiperda.Using the method of co-toxicity coefficient,the ratio with co-toxicity coefficient greater than 120 was successfully screened out.The results showed that the co-toxicity coefficients were greater than 120 at the ratios of chlorfenapyr:indoxacarb=1:5,2:5,1:10 and1:15,which indicated that the mixture of chlorfenapyr and indoxacarb had synergistic effect on insecticidal effect of larvae of S.frugiperda under this ratios.The expression levels of CYP4G15and CYP4L4 were upregulated under the stress of two insecticides,indicate that CYP4G15 and CYP4L4 could be induced by the insecticides of chlorfenapyr and indoxacarb,and the two genes might be involved in the metabolism of chlorfenapyr and indoxacarb.3.The function of CYP4G15 and CYP4L4 were silenced by RNAi technology in S.frugiperda.Ds RNA was synthesized in vitro and injected into the larvae of S.frugiperda.The optimal silencing concentration and the silencing time of the two genes was screened by RT-q PCR after treatment with insecticides.The optimal interference concentrations of ds CYP4G15 and ds CYP4L4 were selected as 2000 ng/μL and 1000 ng/μL respectively.After the injection of dsRNA,LC₂₀treatment with chlorfenapyr and indoxacarb showed almost no silencing effect in the first 12 h.ds CYP4G15 and ds CYP4L4 had the best interference efficiency after 24 h treatment with chlorfenapyr,which were 70%and 67%,respectively.ds CYP4G15 had the best interference efficiency of 78%after 24 h treatment with indoxacarb,and ds CYP4L4 had the best interference efficiency of 62%after 36 h treatment with indoxacarb.After the optimal interference concentration of dsRNA was injected into the larvae of S.frugiperda,Then the toxicity was determined by using chlorfenapyr and indoxacarb in the laboratory.The death data were collected at 24 h and 48 h,the result showed that the lethal middle concentration values of ds CYP4G15 and ds CYP4L4 after 24 h chlorfenapyr treatment were decreased by 3.15 and 1.68 times compared with the control group injected with ds GFP,respectively.After 48 h,the lethal concentration decreased by 2.89 and 2.04 times,respectively.After 24 h of indoxacarb treatment,the lethal concentration values of ds CYP4G15 and ds CYP4L4 were decreased by 2.19 and 1.5 times compared with the control group injection of ds GFP,respectively.after 48 h,the lethal concentration values were decreased by 3.48 and 1.06 times,respectively.By comparing the mortality of each treatment,we found that at the same concentration,the mortality of ds CYP4G15and ds CYP4L4 injection were higher than that ds GFP injection.But after 48 h the injection of dsRNA,the mortality rate the injection ds CYP4L4 was lower than that ds GFP injection under the stress of indoxacarb.Combined with the change of lethal medium concentration values and mortality rate,it was concluded that injection ds CYP4G15 and ds CYP4L4 could increase the susceptibility to the two insecticides,chlorfenapyr and indoxacarb.