Mechanism Of Aryl Hydrocarbon Receptor(AhR) Signaling Pathway Regulating The Sensitivity Of The Locusta Migratoria To Chlorpyrifos

Aryl hydrocarbon receptor(AhR)is a ligand-binding transcription factor in the cytoplasm,which is mainly involved in the metabolic reaction of toxic substances.In recent years,studies have shown that the transcription factor AhR is involved in the formation of insect resistance to various insecticides.In this experiment,we using Locusta migratoria as experimental material and using RNAi technology and based on the transcriptome data of the Locusta migratoria,the metabolic and detoxification regulation mechanism of the Lm AhR gene of the Locusta migratoria was explored,Providing new methods and new ideas for the development of new targets for pest control and new insecticides.The main contents of this study are as follows:1.Interfering with the Aryl hydrocarbon receptor(Lm AhR)affects the sensitivity of the Locusta migratoria to insecticidesIn this study,the full-length sequence of Lm AhR gene was first cloned;sequence analysis and structural analysis of protein were performed by basic bioinformatics techniques.Finally,the Lm AhR was silenced by RNAi technology to detect the sensitivity of the Locusta migratoria to four typical insecticides.The results showed that the AhR protein consists of the b-HLH domain,the PAS protein domain and the transcriptional activation domain from the N-terminus to the C-terminus.Phylogenetic analysis indicated that the migratory Lm AhR first aggregated with the Lepidopteran AhR;Silencing the Lm AhR increased the sensitivity of the Locusta migratoria to chlorpyrifos,and speculated that the transcription factor AhR may be involved in the metabolic detoxification function of the Locusta migratoria on chlorpyrifos.The above studies provide a theoretical basis for elucidating the function of the Lm AhR and the new control methods of the oriental migratory locust,and also enrich the content of insect resistance research.2.The comparative transcriptome analysis of the Locusta migratoriaIn order to further explore the mechanism of metabolic detoxification of the scorpion transcription factor AhR,a deep-sequenced transcriptome database was constructed.The 47,429 GB of Clean Data was obtained by sequencing;The total 79,429 Unigenes were obtained by assembly,The total length of the sequence was 71,915,521 bp,the N50 Length was 1876 bp,and the Mean Length was 90,560 bp.Using the EDGE tool,refer to the difference of?2 times and p?0.05 as the cutoff value,the differentially expressed genes between the treatment group and the control were selected.Based on the standard,212 genes were differentially expressed,of which 67 genes were down-regulated and 145 genes were up-regulated.Based on the research purpose,we focus on the down-regulated group genes,and further perform egg NOG function annotation,clustering and enrichment analysis on differentially expressed genes.Finally,RT-q PCR was used to verify the transcriptome data.Based on the sequencing analysis,we screened a typical detoxification enzyme gene Lm GSTd7.Therefore,it is speculated that Lm AhR may participate in the metabolic detoxification of the Locusta migratoria by affecting the expression of the Lm GSTd7 gene.3.Sensitivity mechanism of Lm GSTd7 gene to chlorpyrifosBased on the above transcriptome analysis results,this chapter further explores the metabolic response of GSTD7 detoxification enzymes to four insecticides.Firstly,RT-q PCR was used to analyze the expression patterns of Lm AhR and Lm GSTd7 genes in different tissues and different developmental stages of the Locusta migratoria.The results showed that the Lm AhR and Lm GSTd7 genes were expressed in the tissues examined,and the tissue expression profiles of Lm GSTd7 and Lm AhR genes were similar,and their expression levels in the brain and hemolymph were relatively high;The Lm AhR gene has the highest expression in the nymphal stage of the first instar,and the higher expression in the nymphal stage of the second to fourth infestation,While Lm GSTd7 has the highest expression in the egg stage,andthe expression level in the third and fifth instar nymphs is relatively high.The Lm GSTd7 gene was silenced by RNAi technology to detect the sensitivity of the Locusta migratoria to different insecticides.As a result,the sensitivity of the Locusta migratoria to chlorpyrifos was significantly increased after treatment with the four insecticides.It is speculated that the transcription factor AhR is involved in the expression of Lm GSTd7 gene and mediates the metabolism of chlorpyrifos.Lm GSTD7 was expressed in BL21(DE3)by prokaryotic expression technology.After purification by Ni-NTA affinity protein,the GSTD7 protein was successfully obtained,which laid a foundation for subsequent pesticide metabolism experiments in vitro.