Cloning And Functional Verification Of Sodium Channel Gene Of Sitobion Avenae(Fabricius)and Aphis Gossypii Glover

Both Sitobion avenae(Fabricius)and Aphis gossypii Glover belong to the Hemiptera(Sternorrhyncha)suborder(Sternorrhyncha).They mainly damage wheat and cotton,respectively,which reduces the yield and quality of both,causing serious economic loss.At present,the main method on control of aphids is chemical control.Due to pyrethroid insecticides have insecticidal broad spectrum,low toxicity to mammals and other special effects are widely used to control S.avenae and A.gossypii,but also led to its developed resistance to aphids.The target of pyrethroid insecticides is voltage-gated sodium channel.Therefore,the study of sodium channel of aphids helps us to better understand aphids and pyrethroid insecticides interaction between resistance mechanisms.In this assay,we aimed at cloning of the full length of the sodium channel genes of S.avenae and A.gossypii,and analyze of their sodium channel gene sequences,and preliminarily study the methods of successful expression of sodium channel of aphids in vitro and the voltage gating property of accessory protein TipE of the sodium channel.These findings will not only fill the vacancy of aphid sodium channel research,but also help us to discover the resistant mechanism of aphids to pyrethroid insecticides.(1)Cloning of the sodium channel gene sequence of S.avenae:The sodium channel genes SaNavI,SaNavⅡ and SaTipE were cloned by RT-PCR technology(NCBI sequence numbers:MN176137,MN161583 and MN198189).The open reading frame of the SaNavⅠ sequence is 3423 bp in length,encoding 1141 amino acids in total;the open reading frame of SaNavⅡ sequence is 2874 bp in length and encodes a total of 958 amino acids;the open reading frame nucleotide sequence of SaTipE includes 1353 bp,which encodes a total of 451 amino acids.The analysis shows that SaNavI and SaNavⅡ include two homology domains Ⅰ,Ⅱ and Ⅲ,Ⅳ,respectively.Each domain contains 6 transmembrane fragments S1,S2,S3,S4,S5 and S6.Between the Ⅰ-Ⅳ domains S5-S6,there are four amino acid residues D,E,N,S,Ⅲ and Ⅳ between amino acid residues Met-Phe-Met(MFM).The 23 SaNavI and 18 SaNavⅡ clones were sequenced and analyzed by multiple sequence comparisons.The results showed that there were 4 alternative splicing types(exon j,x,y and b)in the sodium channel gene of S.avenae.and a mutex k/1.SaNavI and SaNavⅡ have 11 RNA editing sites.There is two pairs of linkage sites M915-F1017 and L915-Y1017 in SaNavI.The research results are as follows:(2)Cloning of the sodium channel gene sequence of A.gossypii:The sodium channel genes AgNavI,AgNavII and AgTipE(NCBI sequence numbers:MN689724,MN685725 and MN966969)were cloned using RT-PCR technology.The AgNavI gene sequence includes 3447 bp open reading frame,coding 1149 amino acids in total;The sequence of AgNavⅡ gene includes a 2874 bp open reading frame,which encodes a total of 958 amino acids;the open reading frame nucleotide sequence of AgTipE includes 1353 bp,which encodes a total of 451 amino acids.After analysis,the two amino acid sequences of AgNavI and AgNavⅡ encoding the two ends of the gene include 4 homology domains Ⅰ,Ⅱ,Ⅲ and Ⅳ AgNavI includes homology domains Ⅰ and Ⅱ,and AgNavI includes homology domains Ⅲ and Ⅳ.Each domain includes 6 transmembrane fragments,namely S1,S2,S3,S4,S5 and S6.The polypeptide chain between S5 and S6 of I-Ⅳ homology domains contains D,E,N,S.The polypeptide chain connecting the homology domain Ⅲ and the homology domain Ⅳ contains the MFM module.The sequencing results of 23 ANavI and 18 AgNavⅡ were compared and analyzed for multiple sequences.There are 3 alternative splicing types in A.gossypii,namely exon j,exon z and exon b.There are 4 RNA editing edits in AgNavI and AgNavⅡ.There is also two pairs of linkage sites M915-F1017 and L915-Y101 7 in AgNavI.(3)Functional Verification:The functional verification was carried out by using the cDNA of theα subunit of the sodium channel of S.avenae and its subunit SaTipE cDNA and the DmNavl cDNA in Xenopus oocytes.M915L and Y1017F linkage sites have been found in both SaNavⅠ and AgNavⅠ.In order to explore its functionality,we obtained mutants of M915L and Y1017F by site-directed mutagenesis of the sodium channel of D.melanogaster to expressed in Xenopus oocytes.At present,experiments have found that M915-F1017 records fast current and Ⅰ-Ⅴ current,while L915-Y1017 only records weak current.Electrophysiological results show that both SaTipE and DmTipE can increase the expression of DmNav1;When SaNavⅠ and SaNavⅡ are co-expressed with the SaTipE in oocytes,the results show that although the current is weak when the accessory protein are co-expressed,it can still increase the expression of the sodium channel of S.avenae.From this,it can be seen that the TipE of the sodium channel of the two insects has a significant increase in the expression of the sodium channel.(4)Homology comparison:SaNavⅠ and SaNavⅡ,AgNavⅠ and AgNavⅡ,RpNavI and RpNavⅡ(NCBI sequence number:XP026806616 and XP026806613)sodium channel gene of Rhopalosiphum padi and DmNav(NCBI sequence number:AAB59195)were subjected to ’multiple sequence alignments.It was found that the amino acid sequence homology between SaNavⅠ and DmNav was 66.7%,and the homology between SaNavⅡ and DmNav was 66%.SaNavⅠ and AgNavI were the most similar at about 99.90%,followed by MpNavI at 97.81%,and the similarity rates of RpNavI and SaNavⅠ reached 97.39,respectively.%;SaNavⅡ is the most similar to AgNavⅡ at 99.82%,followed by RpNavⅡ at 97.90%,and MpNavⅡ at 96.43%.