The Molecular Mechanism Of MicroRNA Regulates P450 Gene Expression Mediating Resistance To Imidacloprid In Sitobion Miscanthi

Sitobion miscanthi is a major agricultural pest that harms wheat crops mainly by feeding on plant juice,secreting honeydew and spreading plant viruses.Imidacloprid is a nicotinic chloride agent with high toxicity to aphids and other stinging insects.However,with the long-term application of imidacloprid in a wide range of areas,many pests developed serious resistance.Cytochrome P450 mediated metabolic enhancement is an important reason for the development of resistance to imidacloprid.In this study,transcriptional sequencing technology was used to screen out miRNAs and P450 genes that were differentially expressed in aphid imidacloprid resistant strains and sensitive strains of S.miscanthi,and the correlation between miRNAs and P450 genes was analyzed using miRanda and RNAhybrid software,The role of differentially expressed P450 genes in imidacloprid resistance was verified by RNAi technology.Then,A series of experiments,including q RT-PCR,Dual-Luciferase Reporter,and feeding of miRNA agomir/antagomir in vivo,were conducted to further investigate the interaction between miRNA and P450,and finally to clarify the molecular mechanism of miRNA regulation of resistance to imidacloprid mediated by P450 gene expression.The main research results are as follows:1.Determination of resistance level and synergistic effect of S.miscanthi.to ImidaclopridA high resistance strain of imidacloprid with a resistance ratio of 44.33 times was obtained by testing the toxicity of S.miscanthi against imidacloprid after 25 successive generations in laboratory with dipping method.The synergistic test showed that PBO had the highest synergistic multiplier of 8.03 on imidacloprid,while TPP and DEM had poor synergistic multiplier of 4.05 and 1.64,respectively.2.Screening of differentially expressed miRNAs and their target genes of S.miscanthi.A total of 172 miRNAs were identified by transcriptomic sequencing of imidacloprid resistant and sensitive strains of S.miscanthi.Among them,24 miRNAs were down-regulated in imidacloprid-resistant strains and 29 were up-regulated in imidacloprid-resistant strains.The expression of 19differentially expressed miRNAs in imidaclopride-resistant and sensitive strains was determined by q PCR,which was consistent with the transcriptomic sequencing results.These miRNAs may be associated with imidacloprid resistance.Then the target genes of miRNAs were predicted,and some miRNAs could bind to corresponding target genes.3.Correlation analysis between miRNA and P450 gene.The 3’UTR region of CYP6CY6 and CYP6CY8 genes was obtained by RACE.Upload the sequence to the Genbank database.The CYP6CY6 with 1527bp open reading frame（ORF）and 121bp3’UTR,and the entry number was OP957415.CYP6CY8 with a 5’UTR of 36bp,an open reading frame（ORF）of 1539bp,and a 3’UTR of 463bp,with entry number OP957416.Through the software（miRanda,RNAhybrid）,The binding site of miRNA-3047 was located at the 3’UTR 23-46bp of CYP6CY6 gene.The binding site of miRNA-3037 was located at CYP6CY8 gene 3’UTR 338-359bp.4.The role of CYP6CY6 and CYP6CY8 genes in imidacloprid resistance of S.miscanthiThe role of CYP6CY6 and CYP6CY8 genes in the resistance to imidacloprid of S.miscanthi was verified by q PCR and RNAi experiments.q PCR results showed that CYP6CY6 and CYP6CY8 gene expressions in imidacloprid resistant strains were significantly up-regulated,which were 1.62 and 3.82times of those in sensitive strains,respectively.RNAi experiment results showed that after feeding ds RNA for 24h,the silencing efficiency of CYP6CY6 and CYP6CY8 genes reached 33.57%and 46.65%,respectively,compared with the control（ds GFP）.After feeding ds RNA for 24h,the sensitivity of S.miscanthi was measured,and the mortality rate increased significantly,which was 2.00 and 2.37 times that of the control group（ds GFP）.These results indicate that CYP6CY6 and CYP6CY8 genes play an important role in the resistance of S.miscanthi to imidacloprid.5.Regulation of miRNA-3047 and miRNA-3037 on CYP6CY6 and CYP6CY8 gene expression The effects of miRNA-3047 and miRNA-3037 on the expression of CYP6CY6 and CYP6CY8 genes was studied by a series of experiments including Dual-Luciferase Reporter,q RT-PCR,and feeding of miRNA agomir/antagomir in vivo.The results of the Dual-Luciferase Reporter showed that the recombinant plasmid pmir GLO-CYP6CY6 UTR and pmir GLO-CYP6CY8 UTR co-transfected miR-3047-agomir and miR-3037-agomir,respectively,significantly inhibited the luciferase activity of firefly.These results indicate that miR-3047 and miR-3037 can bind to the 3’UTR region of CYP6CY6 and CYP6CY8 and regulate the expression of CYP6CY6 and CYP6CY8 genes,respectively.After feeding miR-3047 and miR-3037 agomir for 24 h,the CYP6CY6 and CYP6CY8 genes decreased by 82.2%and87.5%,respectively,while the mortality rate of S.miscanthi was increased by 60.0%and 36.7%after treatment with imidacloprid LC₅₀for 24 h.On the contrary,after feeding miR-3047 and miR-3037antagomir for 24 h,the CYP6CY6 and CYP6CY8 genes increased by 1.5 and 3.3 times,respectively,and after treatment with imidacloprid LC₅₀for 24 h,the mortality rate of S.miscanthi decreased by 37.0%and66.7%.In conclusion,miRNA-3047 and miRNA-3037 reverse-regulate the expression of CYP6CY6 and CYP6CY8 genes,respectively,and mediate the resistance of S.miscanthi to imidacloprid.