Cloning Of Ryanodine Receptors From Three Insects And Comparative Analysis Of Their Sensitivity Differences To Chlorantraniliprole

Ryanodine receptor(RyR)is the largest calcium channel and plays a critical essential role in the whole life of insects.RyR is the main target of anthranilic and phthalic diamide insecticides such as chlorantraniliprole,flubendiamide,and cyantraniliprole.These pesticides shows extremely high activeities to Lepidopteran and part of Coleopteran insect species,but safe to other insects,mammals and fishes.Some research gaps existing in the mode of action of anthranilic and phthalic diamide insecticides must be addressed.Among these gaps four are important.(1)Since the RyR genes are very big in size,it is not easy to get the full ORF sequences.Therefore,the RyR genes in many important pest insects remain unidentified.(2)The conclusion that RyRs act as the target of anthranilic and phthalic diamide insecticides has only been confirmed in Dipteran and Lepidopteran insect species.(3)The crystal structure of insect RyR has not been reported up to now.(4)The reason of specific activity of to anthranilic and phthalic diamide Lepidoptera insects is unknown either.In the present study,we first cloned RyR genes in three agricultural pests Sogatella furcifera RyR(SfRyR),Leptinrotarsa decemlineata RyR(LdRyR)and Helicoverpa assulta RyR(HaRyR).We then prove that SfRyR and LdRyR acted as chlorantraniliprole targets using RNAi methods.Moreover,the HaRyR alternative splicing isoforms were analysized in Helicoverpa assulta.Finally,using phylogenetic tree,homology model and molecular docking method,we analyzed the possible reasons that the three insects showed sensitivity differences to chlorantraniliprole.1.RNAi mediated knockdown of the ryanodine receptor gene decreases chlorantraniliprole susceptibility in Sogatella furciferaWe isolated a 15985 bp full-length cDNA(named SfRyR)from Sogatella furcifera,a serious rice planthopper pest throughout Asia.SfRyR encodes a 5140-amino acid protein,which shares 78%-97%sequence identities with other insect homologues,and less than 50%identities with H.sapiens RyRl-3.All hallmarks of the RyR proteins are conserved in SfRyR.In the N-terminus,SfRyR has a MIR domain,two RIH domains,three SPRY domains,four copies of RyR repeated domain and a RIH-associated domain.In the C-terminus,SfRyR possesses two consensus calcium ion-binding EF-hand motifs,and six transmembrane helices.Temporal and spatial expression analysis showed that SfRyR was widely found in all development stages including egg,first through fifth instar nymphs,macropterous adult females and males.On day 2 fifth-instar nymphs,SfRyR was ubiquitously expressed in the head,thorax and abdomen.Dietary ingestion of dsSfRyRl and dsSfRyR2 significantly reduced the mRNA level of SfRyR in the treated nymphs by 77.9%and 81.8%respectively,and greatly decreased chlorantraniliprole-induced mortality.Thus,our results suggested that gene encoded a functional RyR that mediates chlorantraniliprole toxicity to S.furcifera.2.RNAi suppression of the ryanodine receptor gene results in decreased susceptibility to chlorantraniliprole in Leptinotarsa decemlineataWe cloned and characterized a 15792-bp full-length LdRyR cDNA that encoded a 5128-amino acid protein.LdRyR shares 85%-92%amino acid similarities with other insect RyR homologues,and 59%-61%similarities with those from Caenorhabditis elegans and Homo sapiens.All hallmarks of the RyR proteins are conserved in LdRyR.LdRyR has a MIR domain,two RIH domains,three SPRY domains,four copies of RyR domain and a RIH-associated domain in the N-terminus,and it possesses two consensus calcium ion-binding EF-hand motifs and six predicted transmembrane helices in the C-terminus.Temporal,spatial and tissue-specific expression patterns of LdRyR were evaluated.LdRyR expression level was increased constantly from egg to wandering stages,dropped in pupal stage and was increased again in the adult stage.It was widely expressed in the head,thorax and abdomen of day 3 fourth-instar larvae.Moreover,it was ubiquitously expressed in all inspected tissues including epidermis,foregut,midgut,ileum,rectum,fat body,ventral ganglia and Malpighian tubules in day 3 fourth-instar larvae.Dietary introduction of double-stranded RNA of LdRyR significantly reduced the mRNA levels of the target gene in the larvae and adults respectively,and significantly decreased chlorantraniliprole-induced mortalities.Thus,our results suggested that LdRyR encoded a functional ryanodine receptor in L.decemlineata.3.Cloning,gene structure and alternative splicing analyses of Helicoverpa assulta RyR geneThe full length of Helicoverpa assulta ryanodine gene cDNA(HaRyR)was obtained.Its ORF is 15414 bp and encodes 5137 amino acids.HaRyR shares 79%-99%amino acid similarity with orther insect RyRs.It contains all hallmarks of conserved domains in RyRs.In the N terminus,there are one MIR domain,two RIH domains,three SPRY domains,four RyR domain and one RIH-associated domain.In the C terminus,two EF-hand motifs and six transmembrane helices are present.Compared with the Helicoverpa assulta genome database,HaRyR gene includes 110 exon,whose length ranges from 18 to 326 bp.Moreover,4 alternative splicing sites were identified,they located on the exon 32,66,67 and 95 respectively.This resulted in a total of 9 splicing isoforms.Furthermore,the expression of these splicing isoforms was evaluated in different development stages(egg,Ith instar,3th instar,5th instar,pupa and adult),by multi clone sequencing and RT-PCR band gray value.4.Phylogenetic analysis and identification of binding sites to chlorantraniliprole in three insect RyRsWe collected 27 RyRs origined from in 23 species and constructed a phylogentic tree.The RyR proteins are conserved at the amino acid level during the long term evolution in insecta and mammal category.The SfRyR,LdRyR and HaRyR belonged to Hemiptera,Coleoptera and Lepidoptera clades respectively.Moreover,the gene structures of RyRs from 9 insects in 5 orders were analyzed.Exon numbers are conserved in the same order,but significantly different between orders.The phenomenon indicate the RyRs are under high selective pressure.By homologous modeling,three-dimensional(3D)structures of three RyRs and C terminus were predicted.Moreover,molecular docking was performed to determine the binding mode for chlorantraniliprole.The hydrogen bonding and pi-pi interactions were fully examined.The structural differences of binding pockets for chlorantraniliprole among 3 RyRs suggest their sensitivity differences to chlorantraniliprole among the three important agricultural pests.