Identification,Screening And Functional Study Of ATP-binding Cassette (ABC) Transporter Genes Involved In Deltamethrin Resistence In The Malaria Vector Anopheles Sinensis

ATP-binding cassette(ABC)transporters are a large group of membrane proteins widely found in archaea,bacteria and eukaryotes.ABC transporters can participate in the absorption,accumulation and excretion of a variety of endogenous and exogenous toxic substances.They play an important role in the process of tissue defense,and are closely related to the resistance of animals or insects.Anopheles sinensis is an important vector of malaria and malayan filariasis,and widely distributes in East Asia and Southeast Asia.Due to the widespread use of pyrethroid insecticides(especially deltamethrin),the resistance of An.sinensis to these drugs has become increasingly serious and has become a major obstacle to mosquito control.Nowadays,little information is known for ABC transporter and its resistance to deltamethrin in An.sinensis.Therefore,in the present study potential ABC transporter genes were identified in An.sinensis genome data by bioinformatics analysis,and their nomenclature,domain,gene structure,scaffold distribution and phylogeny were studied.The ABC transporters genes involved in deltamethrin resistance were screened by RNA-seq and qPCR,and the gene function was further investigated based on RNAi,ATPase activity assays and molecular docking.The main contents of this paper are as follows:(1)61 ABC transporter genes were identified in An.sinensis based on the genomic data of An.sinensis.They were divided into eight subfamilies,and have various domains,including whole,incomplete,semi-molecule and a quarter of ABC transporters.The ABCG subfamily,containing 21 members,is the largest one in the ABC subfamily,and the ABCE subfamily,containing only one ABC transporter,is the least abundant subfamily.Phylogenetic analysis showed that ABCB,ABCD,ABCE,ABCF and ABCH were more conservative subfamilies in An.sinensis,and their genes were homologous with those of An.gambiae and Drosophila melanogaster.Moreover,ABCC and ABCG subfamilies have experienced gene duplication events.(2)The expression levels of all ABC transporter genes were analyzed and compared using RNA-seq in three field deltamethrin resistant populations(AH-FR,CQ-FR and YN-FR)and one laboratory sensitive strain(WL-LS).The results showed that six genes(AsABCG28,AsABCA5,AsABCC9,AsABCC11,AsABCG7 and AsABCG23)were significantly differently expressed in at least one resistant population.Among them,AsABCG28 was the only gene that was significantly up-regulated in all three populations.The results of qPCR showed that three ABCB genes(AsABCB1,AsABCB3 and AsABCB5)was significantly up-regulated expression after 6 hours of deltamethrin treatment,and AsABCG28 gene was significantly up-regulated expression at 12 and 24 hours after treatment.(3)AsABCG28-dsRNA was synthesized in vitro by T7 kit and introduced into 2-day-old An.sinensis larvae by feeding method.The results of qPCR showed that the expression level of AsABCG28 gene was significantly decreased,and the silencing of AsABCG28 gene could significantly increase the lethality of deltamethrin-induced larvae of An.sinensis.(4)The recombinant plasmid AsABCG28/pPICZA was constructed,linearized and electrotransformed into Pichia pastrois GS115.High-copy recombinant was obtained by screening of Zeocin antibiotics.After 24 hours of methanol-induced expression,the target product was purified by tissue grinding,ultracentrifugation and nickel affinity chromatography.As a result,1 liter fermentation broth can harvest 0.1 mg protein AsABCG28 with ATPase activity of 25 nmol/mg/min.Within the concentration range(0-1 mM),deltamethrin did not induce ATPase activity of recombinant protein AsABCG28,while 3-phenoxybenzoic acid,a metabolite of deltamethrin,significantly induced ATPase activity in dose-dependent manner.The results of molecular docking showed that 3-phenoxybenzoic acid could bind well to the TMD of AsABCG28 with a free energy of-29.4036 kcal/mol,and the hydroxyloxy group of 3-phenoxybenzoate formed a hydrogen bond with the residue Val431 of AsABCG28 with the bond length of 2.8 ?.In conclusion,the present study provides a basic information framework for understanding ABC transporter subfamily of genes,and lays an important foundation for better undertanding and in-depth study of the role of ABC transporter genes in deltamethrin resistance.The AsABCG28 gene is closely related to the deltamethrin resistance of An.sinensis,mainly by transporting 3-phenoxybenzoic acid,a deltamethrin metabolite,to reduce its accumulation in the body,thereby inceasing the resistance of the mosquito to deltamethrin.