Function And Transcriptional Regulation Of CYP4G Subfamily Genes In Integument Development Of Mythimna Separata

The oriental armyworm,Mythimna separata（Walker）,is a major crop pest in China,causing huge losses to crops.However,with the nonstandard use of insecticides,the re-sistance of oriental armyworm is increasing,and the development of green and harmless new insecticides has become a research hotspot.It has been reported that CYP4G subfamily（CYP4Gs）genes in the P450 family are involved in the synthesis of integument hydrocar-bons.Based on this research background,RT-q PCR,RNAi,5’-RACE and double luciferase reporter system were used to study the CYP4Gs genes of oriental armyworm,and explore the function and transcriptional regulation mechanism of CYP4Gs genes in larvae integu-ment,so as to provide a theoretical basis for better development of new pesticide targets.The main research results are as follows:1.Cloning of three CYP4G subfamily genesThe cDNA sequences of CYP4Gs genes were cloned and named as CYP4G199,CYP4G200 and CYP4G201,respectively.The length of open reading frame（ORF）was about 1600 bp,encoding about 550 amino acids.Helix C,Helix I,Helix K,PERF and Heme-binding domain are conserved domains of P450.Genome structure analysis found that CYP4G199 in CYP4G201 longest intron sequence,known as nested genes.The phylogenetic tree of CYP4Gs genes was constructed,and two branches of differentiation were found.CYP4G199 and CYP4G201 belonged to CYP4G1 type,and CYP4G200 belonged to CYP4G15 type.2.Temporal and spatial expression pattern of CYP4G subfamily genesThe expression patterns of CYP4Gs genes in different developmental stages of oriental armyworm were detected by RT-q PCR.The results showed that CYP4G199 was highly ex-pressed in the first instar larvae and male adults,CYP4G200 was highly expressed in the first instar larvae,and CYP4G201 was highly expressed in the egg stage.The expression of CYP4Gs genes gradually decreased with the increase of larval instar;the expression levels of male adults were higher than those of female adults.CYP4G200 was highly expressed in the head of 4^th instar larvae in different tissues;CYP4G199 and CYP4G201 were highly expressed in integument.CYP4Gs genes was expressed in the integument of different instar larvae,while the expression levels of CYP4G199 in the integument of the fourth instar larvae,CYP4G200 in the integument of the first instar larvae,and CYP4G201 in the integument of the sixth instar larvae were higher than those in the integument of other instar larvae.In the molting process of 3^rd to 4^th instar larvae,the expression level of CYP4G199 at 6 h was significantly lower than that at 6 h.On the contrary,the expression levels of CYP4G200 and CYP4G201 at 6 h were significantly higher than that at 6 h.There was a negative correlation between CYP4G199 and CYP4G201 expression pattern,which was related to CYP4G199being a nested gene.3.CYP4G subfamily genes involved in the integument formation of oriental armyworm larvaeThe ds RNA was injected into the larvae of oriental armyworm by RNAi.Compared with the control group,the CYP4Gs genes expression levels were down-regulated to varying de-grees,including 50%down-regulation of CYP4G199,48%down-regulation of CYP4G200and 72%down-regulation of CYP4G201.From the phenotype after gene silencing,CYP4Gs genes were involved in the formation of oriental armyworm integument,and CYP4Gs genes silencing led to the absence of larval integument.At the same time,according to the pheno-types presented in different parts of the injection,it showed that there were differences in the parts where they played a role.CYP4G199 and CYP4G201 were mainly involved in the formation of larvae integument,and CYP4G201 had important physiological functions in the head of larvae.4.FTZ-F1 is involved in the regulation of CYP4G subfamily genesUsing 5’-RACE technology,the transcription start site of CYP4Gs genes was identified as adenine nucleotide（A）.Through the detection of dual luciferase reporter system,it was found that CYP4G199 had key elements regulating expression between-292 and+24 and CYP4G201 between-214 and+39.Bioinformatics software PROMO and Jaspar were used to predict the above regions and the regions between-465 and+86 of CYP4G200,and it was found that there were sites binding to transcription factor FTZ-F1.RT-q PCR was used to analyze the temporal and spatial expression distribution of FTZ-F1 in oriental armyworm.The results showed that the gene was highly expressed in the 4^th and 5^th instar larvae,and the expression level in male adults was slightly higher than that in female adults.In different tissues of 4^th instar larvae,it was highly expressed in brain,followed by fat body,and also in integument and ventral nerve cord;among the integument of the 1^st to 6^th instar larvae,the expression was most obvious in the integument of 6th instar larvae;during the molting process of 3^rd to 4^th instar larvae,the expression was obvious at 9 h.After silencing FTZ-F1by RNAi,the relative expression level was down-regulated by 79%.At the same time,the expressions of CYP4G199,CYP4G200 and CYP4G201 were also down-regulated by 50%,48%and 72%,respectively,and the incomplete integument of larvae during molting was observed,and even the death caused by difficulty in integument formation.The above results showed that CYP4Gs genes were involved in the formation of oriental armyworm integument,and transcription factor FTZ-F1 might be involved in the regulation of CYP4Gs genes.It regulates the expression of CYP4Gs genes by binding to the upstream promoter of CYP4Gs genes,thereby affecting the formation of oriental armyworm integu-ment.However,the differences in transcriptional regulation of CYP4Gs genes in oriental armyworm need further experimental studies.