The Preliminary Study Of RSC1 Ion Channel Gene And The Sodium Channel Auxiliary Subunit Gene Of Rhopalosiphum Padi

The bird cherry-oat aphid,Rhopalosiphum padi?Hemiptera: Aphididae?,a serious pest in the wheat filed,damaged the wheat by sap-sucking or by transmitting with a serious virus?barley yellow dwarf virus,BYDV?which resulted in enormous economic reduction of wheat production annually in the world.Pyrethroids are important synthetic insecticides which were widely used due to it is low toxicity for mammals.However,the extensive application of Pyrethroids has contributed to the development of insecticide resistance in the target pests.Analysis of new potential neural targets of insecticide is important for insecticide resistance management.In this study,we cloned the full-length sequences of RSC1 gene and five voltage-gated sodium channel auxiliary subunit genes from R.padi.The bioinformatic characteristics and the expression patterns of the genes under the ?-cyhalothrin induction as well as in different development stages of R.padi were investigated.The results of the study provide a foundation for the further research of these genes.1.Molecular cloning and bioinformatics analysis of RSC1 gene in R.padiRT-PCR was used to clone the full ORF cDNA of RSC1 gene from R.padi.The GenBank accession number of the gene is KU640190.The open reading frame length is 6687 bp,which encodes 2228 amino acids.NCBI Blastp results indicate that the amino acid sequences of RSC1 shows high identity to the corresponding protein from Acyrthosiphon pisum,with more than 90% identifies.Structural analysis by TMHM-M-2.0 software shows that the RSC1 has the typical structural feature of SC1 channel,which is composed of four homologous domains,each containing six transmembrane segments.Multiple sequence alignments of the DSC1 protein with the orthologs from nine other insects species including fruit fly?Drosophila melanogaster?,crockroach?Blattella germanica?,honeybee?Apis mellifera?,red flour beetle?Tribolium castaneum?,Indian jumping ant?Harpegnathos saltator?,bumblebee?Bombus terrestris?,Florida carpenter ant?Camponotus floridanus?,leaf-cutting ant?Acromyrmex echinatior?and pea aphid?Acyrthosiphon pisum?,illustrates that RSC1 and its orthologs share the unique sequence feature of a DEEA motif in the pore regions.A phylogenetic tree of SC1,Na V and Cav highlights the SC1 channel family is evolutionarily closer to the voltage-gated sodium channel family next to calcium channel family.Moreover,the SC1 have been found in invertebrates but not in human being and vertebrates,these channels could be used as neurotarget to exploit novel pesticides that are human being and vertebrates.2.The expression patterns of R.padi RSC1 gene after exposure to ?-cyhalothrin insecticideqRT-PCR was used to characterize the expression of RSC1 in R.padi adults which were exposed to LC15?0.1484 mg/L?,LC35?0.3244 mg/L?and LC₅₀?0.5153 mg/L?concentrations of ?-cyhalothrin,respectively.The expression level of RSC1 gene 6 h post-exposure to the three concentrations of ?-cyhalothrin was significantly decreased to 0.57-,0.82-and 0.78-fold that of the control respectively,whereas it increased to 2.19-,1.33-and 1.19-fold that of the control 24 h post-exposure to the chemicals,respectively.The expression level of RSC1 gene significantly increased in R.padi adults that were exposed to LC15 concentration?-cyhalothrin.Therefore,SC1 ion channel would play a important role in sustaining the stability of the neuronal excitability.3.Molecular cloning and bioinformatics analysis of subunits of voltage-gated sodium channel genes from R.PadiRT-PCR was used to clone the full-ORF cDNAs of five voltage-gated sodium channel auxiliary subunit genes?TipE,TEH1,TEH2,TEH3 and TEH4?from R.padi.The respective open reading frame lengths of the five genes are 1380,951,999,1422 and 1542 bp,which encode proteins of 459,299,332,473 and 513 amino acids,respectively.The amino acid sequences of the five genes TipE and TEH1-4 show high identity to the corresponding proteins from Acyrthosiphon pisum with more than 80% identifies,whereas they show low identity to the corresponding proteins of Drosophila melanogaster.The RpTipE is composed of two putative membrane-spanning segments,including the extracellular loop and two intracellular extremities.This topological organization is similar to the calcium ion channel?-subunit?Slo-??of mammals.The phylogenetic tree of Nav auxiliary subunits of vertebrate and insects was separated into two major clusters.One cluster contains the ?-subunit of vertebrate and the second cluster assembles the TipE and TEH1-4 from insects.Specifically,the second cluster was constituted of three sub-branch,TEH3 and TEH4 were clustered in a sub-branch.So subunits of sodium channel of insects would like to be regarded as a potential novel target of new pesticides.4?Expression patterns of R.padi TipE and TEH1-4 genesqRT-PCR was used to characterize the expression of R.padi TipE,TEH1,TEH2,TEH3 and TEH4.The results showed that the five genes were expressed at all developmental stages of R.padi,but the expression varied among different developmental stages.Besides,we treated apterous adult R.padi females with a LC₅₀?0.5153 mg/L?and LC₂₅?0.2544 mg/L?concentration of ?-cyhalothrin over two periods,including 12 hours and 24 hours.Then the expression of TipE-like genes were measured among surviving insects.The results showed that expression levels of TipE-like of five genes down-regulated after exposure to ?-cyhalothrin after 12 hours,and the relative expression level of LC₂₅ group were 0.4135-,0.3181-,0.2194-,0.4084-and 0.2951-fold,respectively.Specifically,and the relative expression level of five genes were significantly different with the control.For the LC₅₀ group,the relative expression level were 0.4135-,0.3181-,0.2194-,0.4084-and 0.2951-fold respectively after 12 h pesticide exposure,especially,the the relative expression level of five genes were significantly different with the control.After 24 h pesticide exposure,the trend of related expression levels of TipE-like of five genes were descending.The expression level of TipE-like genes decreased to 0.5519-,0.4352-,0.3440-,0.1284-and 0.4235-fold that of the control respectively.The expression level of five genes significantly declined in R.padi adults exposed to ?-cyhalothrin at the dosage of LC₂₅.About the LC₅₀ group,TipE-like genes decreased to 0.4881,0.3286,0.3126,0.1166-,0.3816-fold that of the control respectively,after 24 h pesticide exposure.Especially,the the relative expression level of five genes were significantly different with the control.Hence,TipE-like family may play the vital role in voltage-gated sodium channel of insects of their function.