Discovery Of Type ? Sodium Channel Gene Of Tribolium Castaneum And Its Application In Basic Research As A Insecticide Target

The insect voltage-gated sodium channels are the primary targets of pyrethroids and novel efficient insecticides such as indoxacarb and metaflumizone.Gene duplication provides raw genetic material for generating novel eukaryotic genes and causes functional differentiation of duplicated genes.While only one sodium channel gene is found in most of the insect genomes,our studies have found the occurrence of sodium channel gene duplication in the genome of Tribolium castaneum,which generated two genes,TcNavl and TcNav2.On this basis,the full length of TcNav2 was cloned,and the sequence homology,phylogenesis,genomic structure and selective splicing sites of TcNavl and TcNav2 were compared.The spatiotemporal expression patterns of TcNavl and TcNav2 in different tissues and developmental stages were comparatively analyzed by qRT-PCR.We also document the role of TcNavl and TcNav2 in pyrethroids toxicity and.castaneum development by RNA interference.The results will not only assist in understanding the function and evolution of insect sodium channels,but also provide important information for the development of novel insecticides.The main results are as follows:The full-length cDNA sequence of TcNav2 was cloned,and the sequence and structural characteristics of TcNavl and TcNav2 were compared.RT-PCR was used to amplify the entire coding sequence of the TcNav2 cDNAs from.castaneum.The composite TcNav2 cDNA contain 6114 nucleotides with an ORF encoding 2037 amino acid residues.An amino acid sequence alignment shows that TcNavl and TcNav2 shares 86%identity with each other,and are 76.1%and 75.5%identical with D.melanogaster para(AAB59195),respectively.Phylogenetic analysis also showed that TcNavl and TcNav2 were clustered with other insect sodium channel genes.TcNavl and TcNav2 genes are structurally similar,including 4 homologous domains(I-IV),and each domain contains 6 transmembrane segments(S1-S6).The motif DEKA(which functions to determine ion selectivity and is located in the homologous domain S5 and S6 junction ring)is highly conserved in TcNav1(D387,E988,K1492,A1785)and TcNav2(D376,E975,K1483,A1776).The genome structure and selective splicing sites of TcNavl and TcNav2 were compared.Both TcNav1 and TcNav2 have 26 exons and 25 introns.Corresponding to TcNavl,exons 8-9 are combined into single exons in TcNav2;Corresponding to TcNav2,exons 18-19 are combined into single exons in TcNav1.The identity between each pair of exons of the TcNavl and TcNav2 genes was 59.1-98.9%.For all 26 exons,the length of 18 exons is strongly conserved in both TcNavl and TcNav2,the lengths of introns on average are 166 bp and 169 bp.Most introns in both TcNavl and TcNav2 are in agreement with the GT-AG consensus sequence and some are other sequence such as GC-AG.Alternative 3'splice site of exon 1 and alternative 5'splice site exon14 occurs exon skipping,correspond to optional exons j and f of para respectively.Corresponding to optional exons a and i of para was found in all cDNA clones.Introns 25 was found in several cDNA clones(transcripts),indicating the occurrence of intron retention.The expression of duplicated sodium channel genes in different tissues and different developmental stages was determined by RT-qPCR.The results showed that the mRNA levels of TcNav1and TcNav2 were the highest in the head,while there was no significant difference among the ovary,thorax,midgut,Malpighian tubules and fat body.There was significant difference between TcNav1 and TcNav2 gene expression in the head,but no significant difference in the other tissues.The developmental expression pattern revealed that the mRNA levels of TcNav1 in the 1-day-old Larvae were significant higher than the other stages.The mRNA levels of TcNavl were significant higher than TcNav2 in all stages.An effective RNA interference system of duplicated sodium channel genes was constructed.To ensure target specificity of RNAi,specific siRNA targeting TcNav1(siRNA-TcNav1)or TcNav2(siRNA-TcNav2)was injected into 20-day larvae or 1-day pupae.The silencing effects were detected by qPCR at 24h,48h,72h,96h and 120h post-injection.The results showed the transcript levels of TcNavl in the injected larvae were significantly suppressed by 74%at 24h post-injection,while that of TcNav2 were reduced 60%at 72h post-injection.The injections of siRNA-TcNav1and siRNA-TcNav2 into 1-day-old pupae suppressed TcNavl and TcNav2 transcripts by 70%and 86%at 48h and 72h post-injection,respectively.Moreover,both TcNav1-RNAi and TcNav2-RNAi were highly selective in knocking down its target gene,and no cross-interference effect was detected since mRNA levels of the closely related gene remained unaffected.The effects of RNA interference on larval susceptibility to insecticides and growth were compared.To assess whether the reductions of TcNavl and TcNav2 transcript levels by RNAi can lead to a change of insect response to pyrethroids,we injected 20-day larvae with siRNA-TcNav1 and siRNA-TcNav2 and performed the bioassay with two insecticides including deltamethrin and beta-cypermethrin on day 1 and day 3 after the injection.The bioassay results showed that the susceptibilities of siRNA treated larvae to deltamethrin decreased significantly when compared to the larvae treated with buffer.The injections of siRNA-TcNav1 or siRNA-TcNav2 to 20-day larvae significantly delayed the pupation of.castaneum.For the larvae injected with buffer alone(control),74%pupationwere observed after the injection.In contrast,only 42%and 63%of pupation rates were observed when larvae were injected with siRNA-TcNav1and siRNA-TcNav2,respectively.For the adult eclosion,90%eclosion was achieved after 1-day pupae were injected with buffer alone.In comparison,only 75%,and 84%of the normal eclosion rateswere observed when the 1-day pupae were injected with siRNA-TcNav1and siRNA-TcNav2,respectively.