Preliminary Study Of Voltage-gated Sodium Channel Function In Rhopalosiphum Padi

The bird cherry-oat aphid Rhopalosiphum padi(Hemiptera:Aphididae)is a serious pest of wheat in the world.This pest can damage wheats by direct feeding or by transmitting barley yellow dwarf virus(BYDV),which results in enormous economic losses on wheat production annually in the world.Chemical pesticide spraying is the major method for the control of R.padi.Pyrethroid insecticides were widely used to control the aphid.Extensive use of pyrethroids has caused high level resistance of R.padi to this type of insecticide.The voltage-gated sodium channel(VGSC)is the target of pyrethroids,and the mutation of voltage-gated sodium channel gene is the main mechanism of pyrethroid resistance in insects.Traditionally,animals were thought to have only one VGSC gene.However,we found that R.padi owned a unique heterodimeric VGSC encoded by two genes(RpNa_vH1 and RpNa_vH2)in recent work.In this study,we analyzed the types of alternative splicing and the mutation sites of the two VGSC genes from R.padi,and the function of the auxiliary subunits of VGSC genes.The results can provide preliminary knowledge on further function analyses of R.padi VGSC,which is important for design and development of new insecticides.The main findings are as follows:1.Alternative splicing type of the R.padi VGSC genesThe gene structure of R.padi VGSC genes was analyzed by using the genomic data in the public database.The length of RpNa_vH1 genomic sequence was 15058 bp,which was interrupted by 22 introns.The length of RpNa_vH2 genomic sequence was 6798 bp,interrupted by 12 introns.Based on the sequence we cloned by PCR,five splicing variants were identified for RpNa_v H1,revealing 3 alternative spliced exons named as rAS1,rAS2,and rAS3 that was corresponded to the exon j,exon 5,and exon b in DmNa_v in Drosophila molanogaster,respectively.Three splicing variants were identified for RpNa_vH2.Two alternative splicing exons were found in the RpNa_vH2 and were named rAS4 and rAS5,which was correspond to a part of the constitutive exon 21 and a constitutive exon 24 in DmNa_v,respectively.2.Detection of mutations in sodium channel of R.padiBased on the analyses of VGSC sequences from cyhalothrin-resistant strains and susceptible strain of R.padi,four mutation sites(V107A?D263G?M284K?E325G and S531G.)were identified in VGSC genes.D263G and M284K are located on the pyrethroid PyR2 binding region of R.padi VGSC,which were possible contribute to the resistance of pyrothroids.3.Effect of RpNa_vH1 and RpNa_v H2 gene RNAi on the expression levels of the auxiliary subunit genes of voltage-gated sodium channelRNAi and RT-qPCR results showed that knockdown of RpNa_vH1 significantly reduced the expression levels of RpTEH1,RpTEH2,RpTEH4 and RpTipE subunit genes,but the expression level of RpTEH3 gene was no significant difference in treatment group(dsRpNa_vH1 injection)and control group(dsGFP injection).Knockdown of RpNa_vH2significantly reduced the expression levels of RpTEH1,RpTEH2,RpTEH3,RpTEH4 and RpTipE subunit genes in R.padi.4.Effect of RpTipE gene RNAi on the gating properties of R.padi VGSCThe results showed that the gene expression levels of sodium channel genes(RpNa_vH1and RpNa_v H2)and the auxiliary subunit genes(RpTEH1,RpTEH2,RpTEH3 and RpTEH4)were significantly reduced when knockdown of RpTipE.After knockdown of RpTipE gene,the mortality of the R.padi was significantly lower than the control(dsGFP injection and without any injection).These results indicated that RNAi of RpTipE gene significantly decreased the susceptibility of R.padi to cyhalothrin and RpTipE may play a significant role in regulating the gating properties of R.padi VGSC.5.Functional verification of R.padi VGSC auxiliary subunit genes in Xenopus oocytesElectrophysiological experiments showed that injection of DmNa_v22 cRNA into Xenopus oocytes couuld result detectable small Na~+currents,whereas larger Na~+currents can be detected when DmNa_v22 cRNA was co-injected with cRNA of RpTEH2,RpTEH3,RpTEH4,and RpTipE,respectively.The results indicated that the auxiliary subunits TEH2,TEH3,TEH4 and TipE of R.padi can increase the Na~+current of DmNa_v22 and shorten the expression time of DmNa_v22.6.Functional verification of R.padi VGSC in Xenopus oocytesIn this study,we explored the in vitro expression of R.padi VGSC in Xenopus oocytes.The oocytes injected with RpNa_v cRNA did not generate any detectable currents with or without DmTipE or RpTipE.