ABC Transporter Mediates The Metabolism Resistance Mechanism Of Three Insecticides In Nilaparvata Lugens

The brown planthopper(BPH),Nilaparvata lugens(St(?)l),is one of the most destructive pests that seriously threatens the high-quality and safe production of rice.However,due to the unscientific use of chemical insecticides,N.lugens has developed varying levels of resistance to a variety of insecticides.Previous studies have demonstrated that the resistance to sulfoxaflor,nitenpyram and clothianidin were regulated by multiple genes in N.lugens,and ABC transporters played very important roles in the resistance to chemical insecticides in pest.However,the mechanism of its metabolic resistance to insecticides in N.lugens was unclear.In this study,ABC transporter genes were systematically identified,and their function in the metabolic resistance to insecticides were verified.The main research results are as follows:1.Identification of the ABC transporter genes of N.lugensBased on the full-length transcriptome sequencing and PCR technique,a total of 32 ABC transporter genes were identified in N.lugens,which encoded 299-2262 amino acid residues,and all identified genes contained the characteristics of the ABC transporter gene sequence using bioinformatics analysis methods.According to the sequence conservation and phylogenetic analysis of the ABC transporter NBD sequence,the ABC transporter genes can be divided into eight subfamilies from ABCA to ABCH(2 ABC genes in ABCA subfamily,4 genes in ABCB subfamily,and 4 genes in ABCC subfamily,3 genes in ABCD subfamily,1 gene in ABCE subfamily,2 genes in ABCF subfamily,15 genes in ABCG subfamily,1 gene in ABCH subfamily).Obvious homology relationships of ABC transporter genes in each subfamily were found with Laodelphax striatellus,Drosophila melanogaster and Homo sapiens.2.Study on the temporal and spatial expression of ABC transporter genes and their response mode under insecticide stressThe expression patterns of the ABC transporter genes in different developmental stages and different tissues were analyzed by quantitative real-time PCR.The results showed that the ABC transporter genes exhibited time specificity in different developmental stages,most genes were highly expressed in the nymph and adult stages,and the expression levels of Nl ABCA1,Nl ABCG3 and Nl ABCG10 in the egg stage were higher than those in other stages;Several ABC transporter genes were highly expressed in the midgut or Malpighian tube,such as Nl ABCG2,Nl ABCG4,Nl ABCG11,Nl ABCG12,Nl ABCG15.Moreover,the expression of Nl ABCG3,Nl ABCG12 was significantly induced in response to the challenge of insecticides.Adding ABC transporter inhibitors could significantly increase the susceptibility of N.lugens to nitenpyram,clothianidin,chlorpyrifos,isoprocarb,etofenprox and sulfoxaflor,respectively.It was suggested that the ABC transporter might be involved in the detoxification and metabolism of insecticides in N.lugens.3.Functional study of ABC transporter genes involved in three neonicotinoid insecticides resistance in N.lugensThe relative expression levels of 32 ABC transporter genes in the resistant strains and susceptible strains were analyzed by RT-q PCR.It was found that Nl ABCG2,Nl ABCG3,Nl ABCG4,Nl ABCG6,Nl ABCG7,Nl ABCG8,Nl ABCG11 and Nl ABCH1 were significantly upregulated in the sulfoxaflor-resistant strain compared with the susceptible strain;Nl ABCD3,Nl ABCE1,Nl ABCF2,Nl ABCG1,Nl ABCG3 and Nl ABCG6 were significantly upregulated in the nitenpyram-resistant strain;Nl ABCD3,Nl ABCG2,Nl ABCG3 and Nl ABCG14 were significantly upregulated in the clothianidin-resistant strain.Nl ABCG3 was significantly upregulated in three resistant strains,and it was speculated that Nl ABCG3 contributed to the evolution of the resistance to the three insecticides in N.lugens.RNAi experiment further clarified that silencing the Nl ABCG3 gene could significantly restore the susceptibility of N.lugens to sulfoxaflor,nitenpyram and clothianidin,respectively.The results indicated that overexpression of Nl ABCG3 mediated the resistance to three insecticides in N.lugens.4.The expression mechanism of Nl ABCG3 gene regulated by miRNAThe expression of Nl ABCG3 gene was up-regulated significantly by silencing Dicer,AGO and Drosha,which were the key genes in the process of mi RNA synthesis,indicating that mi RNA was involved in the regulation process of Nl ABCG3 gene expression.Moreover,it was found that novel?268 could target the CDS region of Nl ABCG3 gene via mi RNA-target gene prediction software.In addition,dual luciferase reporter assays further confirmed the targeting interaction between novel?268 and Nl ABCG3 gene.Injection of novel?268 mimics could significantly inhibit the expression of the Nl ABCG3 gene and increase the susceptibility to the three insecticides whereas the injection of the novel?268 inhibitor significantly upregulated the expression of the Nl ABCG3 gene,resulting in significant increase of the resistance of N.lugens to the three insecticides.These results indicated that novel?268 can reversely regulate the expression of Nl ABCG3 gene,and then mediate the resistance of N.lugens to sulfoxaflor,nitenpyram and clothianidin,respectively.These findings provided an important theoretical basis for comprehensively revealing the metabolic regulation mechanism of the ABC transporter-mediated of N.lugens to insecticides.