Molecular Mechanism Of Chlorantraniliprole Resistance In Spodop Tera Exigua(H(?)bner)

Ryanodine receptors,which form a class of intracellular calcium channels in insects,is the main target of diamide insecticides.To reveal the molecular mechanisms of diamide insecticide resistance,RyRs genes from six species of agricultural pests were cloned and characterised in the present paper.By comparing the differences in amino acid sequence of resistant lines and sensitive lines,no resistant mutation but several polymorphic residues have been found.However,the expression levels of the resistant line at different developmental stages and in different tissues were significantly higher than sensitive line.At last,abundance of ryanodine genes in resistant and sensitive line genome were analysised.1.Clonging of ryanodine receptor gene from six species of agricultural pestsIn order to investigate the mechanisms for the sensitive difference of six insect species to diamide insecticide,RyRs genes from Spodoptera exigua,Helicoverpa armigera,Chilo suppressalis,Nilaparvata lugens,Laodelphax striatellus and Sogatella furcifera were cloned and compared in this study.The RyRs gene's open reading frames from these insect species are 15354-15408 bp long,encoding predicted RyRs of 5118-5136 amino acids.Those RyRs share highly identity with other insects' RyRs.The RyRs of Lepidoptera and Hemiptera contained different low-complexity region(LCR).2.Identification of gene mutations in RyR of the resistant populationAfter 60 successive generations of selection,the topical LD50 of the selected insects was 246.6-fold greater than the sensitive strain,reveal extremely high levels of resistance.In this research,ten single spodoptera exigua full-length cDNA of ryanodine receptor gene(SeRyR)were cloned,which covered the full coding region of the gene.After sequence alignment of these RyRs,many nucleotide polymorphism sites were detected in PxRyR cDNAs and no mutations were found after compared with sensitive strain,which means that chlorantraniliprole resistance of spodoptera exigua wasn't correlated to amino acid mutations.3.Expression profiles of RyR in different developmental stages and various tissues of spodoptera exiguaAs we didn't found any mutations in the resistance population,mRNA expression levels of RyR in different populations were measured by Real-time quantitative PCR in this research.We found that the ryanodine receptor can express in every developmental stages and tissues.The relative expression levels of RyR in larvae and pupa were higher than adults.As in different tisuses in S.exigua,epidemis had the highest expression level,followed by the Malpighian tubule,while the fat body had the lowest expression.The two strains showed the same expression pattern of RyR in the whole life cycle of S.exigua,but the expression levels of SeRyR were significant different.In the different developmental stages and tissues,the relative expression level of SeRyR in resistance populations were higher than sensitive populaitons,especially in epidemis,which was double expressed compared with the sensitive populations.Combined with the previous research,the quantitative PCR was used to investigate the relative versions between resistance and sensitive populaitons.Based on the above mRNA sequences,two pairs of primers were designed in the 5'and 3'end.The results revealed that relative expression levels in the resistance populations were twice than the sensitive populations.Two different pairs of primers for quantitative PCR far from each other showed the same results,indicating that gene duplication may be occurred in the resistance population.And these results were also support our previous researchers,suggesting the chlorantraniliprole insecticide resistance in field populations of spodoptera exigua may be related with gene duplication.More reliable and feasible method is looking for intensive study,to provide the basis for further understanding of the mechanisms of resistance to chlorantraniliprole.