| The important physiological functions,such as,feeding,salivation,immune response,reproductive development,nervous system and excretion were regulated by the inward rectifying potassium channel(Kir)in insects.Recent studies have shown that the Kir channel is a target for Flonicamid and could be used as a novel insecticide target.In this study,Kir genes of the diamondback moth(DBM),Plutella xylostella was cloned from the Cry1Ac-susceptible strain(DBM1A c-S),the Cry1Ac-resistant strain(DBM1Ac-R)and the field population,and the expression of Kir genes in different strains(populations)was analyzed through real-time fluorescence quantitative PCR to investigate whether the Kir genes was associated with the development of resistance to Cry1 Ac toxin.The results obtained were as follows.1.Five Kirs genes(Pxkir1,Pxkir2,Pxkir3 A,Pxkir3B,Pxkir4)from DBM1Ac-S,DBM1Ac-R and DBM Field were cloned and Pxkir1 was found to have three CDS(Coding sequence)sequences in DBM1Ac-R,with 35 mutations in bases between different sequences.Pxkir3 A gene had two CDS sequences in DBM1Ac-R,with 17 amino acid mutations in the IRK Domain region between different lines(populations).Pxkir3 A gene had two CDS sequences with 80 base mutations between sequences and three amino acid mutations in the IRK Domain between strains(populations).Pxkir4 gene has two CDS fragments in DBM1Ac-R with 32 base mutations between sequences,and three CDS fragments in DBM Field with 43 base mutations between sequences and six amino acid mutation sites in the IRK Domain region between strains(populations).Due to the large number of mutations in the IRK Domain region,the functions of Pxkir1,Pxkir3 A and Pxkir4 expressed in the inwardly rectifying potassium channels of the P.xylostella may differ between sequences.2.Analysis of the expression of Kir genes(Pxkir1,Pxkir2,Pxkir3 A,Pxkir3B and Pxkir4)in DBM1Ac-S,DBM1Ac-R and DBM Field showed that the relative expression of Pxkir1 and Pxkir3 B gene did not differ significantly between DBM1 AcS and DBM1Ac-R strains,but both were significantly higher than DBM field population.The relative expression of Pxkir2 and Pxkir3 A was highest in the DBM1 AcR strain,followed by the DBM1Ac-S and lowest in the DBM Field.The relative expression of Pxkir4 gene was highest in the DBM1Ac-R strain,followed by the DBM field population and lowest in the DBM1Ac-S strain.3.Analysis of the expression of Kir genes in the field populations of the P.xylostella after stressed by low-dose of Lambda-cyhalothrin showed that the expression of Pxkir1,Pxkir2 and Pxkir3 A gene were significantly down-regulated compared to CK,by 53.5%,92.5% and 66.4%,respectively;the relative expression of Pxkir3 B and Pxkir4 gene did not change significantly.The analysis of Kir genes expression in the field population after stressed by low-dose of indoxacarb treatment showed that the expression of Kir genes were all significantly reduced,and the relative expression of Pxkir1,Pxkir2,Pxkir3 A,Pxkir3B and Pxkir4 gene were down-regulated by 90%,39.9%,27.5%,67.2% and 76.5%,respectively.In summary,there were a large number of mutations in the sequences of the Kir genes of DBM1Ac-S and DBM1Ac-R strains,and the expression of Pxkir2,Pxkir3 A and Pxkir4 differed significantly between strains.It is supposed that these genes may play a role in the development of resistance of P.xylostella to Cry1 Ac toxin.The relative expressions of Pxkir1,Pxkir2 and Pxkir3 A gene were significantly downregulated after stressed by low-dose Lambda-cyhalothrin,the expressions of all the 5Kirs genes were significantly down-regulated after stressed by low-dose indoxacarb treatment.It is speculated that the Kir genes may respond by expression downregulation during short duration stress of the agents.This study provides useful information for the study of Kir genes in the P.xylostella,and provides a new basis for exploring the effects of common chemicals in the field on the expression of inward rectifying potassium channel genes. |
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